Poly(ADP-Ribose) Regulates Stress Responses
and MicroRNA Activity in the Cytoplasm
Anthony K.L. Leung,1,3Sejal Vyas,1,2Jennifer E. Rood,1,2Arjun Bhutkar,1Phillip A. Sharp,1,2,* and Paul Chang1,2,*
1Koch Institute for Integrative Cancer Research
2Department of Biology
Massachusetts Institute of Technology, Cambridge, MA 02139, USA
3Present Address: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University,
Baltimore, MD 21205, USA
*Correspondence: firstname.lastname@example.org (P.A.S.), email@example.com (P.C.)
Poly(ADP-ribose) is a major regulatory macromole-
cule in the nucleus, where it regulates transcription,
chromosome structure, and DNA damage repair.
stood. Here, we identify a requirement for poly(ADP-
ribose) in the assembly of cytoplasmic stress
granules, which accumulate RNA-binding proteins
that regulate the translation and stability of mRNAs
upon stress. We show that poly(ADP-ribose), six
specific poly(ADP-ribose) polymerases, and two
poly(ADP-ribose) glycohydrolase isoforms are stress
granule components. A subset of stress granule
proteins, including microRNA-binding Argonaute
family members Ago1–4, are modified by poly(ADP-
a condition when both microRNA-mediated transla-
tional repression and microRNA-directed mRNA
cleavage are relieved. Similar relief of repression
is also observed upon overexpression of specific
poly(ADP-ribose) polymerases or, conversely, upon
knockdown of glycohydrolase. We conclude that
poly(ADP-ribose) is a key regulator of posttranscrip-
tional gene expression in the cytoplasm.
Poly(ADP-ribose), or pADPr, is a macromolecular polymer and
posttranslational modification best known for its functions in
the nucleus (Schreiber et al., 2006). These include DNA damage
repair, chromatin remodeling, and transcriptional regulation
(Krishnakumar and Kraus, 2010). However, increasing evidence
suggests that pADPr also functions in the cytoplasm. For
example, pADPr is required for the structure and function of
the spindle in the mitotic cytoplasm (Chang et al., 2004).
Furthermore, the majority of enzymes regulating pADPr syn-
thesis and degradation are localized to the cytoplasm. These
include two of the three isoforms of pADPr glycohydrolase
(PARG) (Meyer-Ficca et al., 2004) and five pADPr polymerase
(PARP) family members whose cellular localizations are charac-
terized (Juszczynski et al., 2006; Kickhoefer et al., 1999; Liu
et al., 2004; Smith and de Lange, 1999; Yu et al., 2005). Seven-
teen PARPs exist in humans, all defined by a conserved PARP
domain, and are classified as pADPr synthesizing, mono-ADP-
ribose (mADPr) synthesizing, or enzymatically inactive based
primarily on the presence of a triad of histidine, tyrosine, and
glutamate(HYE) thoughttoberequiredforsynthesizing theinitial
mADPr and/or subsequent pADPr subunits (Hottiger et al., 2010;
Kleine et al., 2008).
The ability to synthesize mADPr or pADPr is not a prerequisite
for PARP function. For example, PARP-13/ZAP (zinc finger
by degrading viral RNAs in the cytoplasm (Gao et al., 2002).
PARP-13 proteins either lack a PARP domain (PARP-13.2
isoform) or contain a domain lacking key residues of the HYE
motif (PARP-13.1 contains YYV). Such a lack of catalytic activity
does not rule out the possibility that PARP-13 function requires
ADPr modification by another PARP. Such trans-modification
is common among PARPs from the same subfamily, including
modifications between PARP-1 and PARP-2 (Schreiber et al.,
2002) and between PARP-5a and PARP-5b (Sbodio et al.,
2002). Consistent with PARP-13’s role in regulating RNA in the
cytoplasm, pADPr’s synthesizing and degrading activities were
previously identified in cytoplasmic mRNA-protein complexes
(mRNPs) (Elkaim et al., 1983; Thomassin et al., 1985). In this
study, we discover two functions of pADPr in the cytoplasm—
modulating the expression of microRNA (miRNA) targets
and assembling mRNP-rich structures called stress granules
via translational inhibition and mRNA degradation (Fabian et al.,
2010). Despite the diverse fates of mRNA targets, all processes
are mediated through the miRNA-binding protein Argonaute
(Ago1–4 in humans). Recent data suggest that the activities of
miRNAs can be modulated by multiple mechanisms, including
posttranslational modifications of Argonaute (Qi et al., 2008;
Ru ¨del et al., 2011; Zeng et al., 2008), association with other
SGs assemble upon stalled translation initiation (Anderson
and Kedersha, 2008), which can be triggered via two pathways:
(1) phosphorylation of initiation factor eIF2a, which commonly
Molecular Cell 42, 489–499, May 20, 2011 ª2011 Elsevier Inc. 489
occurs during cell stresses, such as heat shock or arsenite-
mediated oxidative stress, or (2) by addition of translation initia-
tion inhibitors that do not involve the phosphorylation of eIF2a.
Here, we show that pADPr is important for SG assembly and
identify six PARPs including PARP-13 that function in these
processes. Interestingly, the majority of these PARPs also bind
to Ago2, suggesting that they have overlapping activities in
miRNA and SG function.
Poly(ADP-Ribose) Localizes to Stress Granules
in the Cytoplasm
Previous work by us and others identified binding interactions
between pADPr and cytoplasmic RNA-binding proteins (Chang
et al., 2009; Gagne et al., 2008); included among these are SG
components. To determine whether pADPr is present in SGs,
cells were stressed with arsenite then stained with antibodies
generated against pADPr (LP96-10) and SG marker eIF3. Four
human cell lines were analyzed: retinal primary epithelial cells
(RPE-1) and three cancer lines, 293T, U2OS, and HeLa (Figures
1A and S1A). In each case, pADPr was enriched in SGs. To
confirm, five additional anti-pADPr antibodies were tested (Fig-
ure S1B and data not shown). This panel of antibodies includes
monoclonal antibody 10H specific for polymers containing
R20 ADP-ribose subunits (Kawamitsu et al., 1984). Each anti-
body stained the interphase cytoplasm and nucleus in untreated
cells and demonstrated a clear colocalization with eIF3-positive
puncta in the cytoplasm upon stress, regardless of fixation
method (methanol or paraformaldehyde; data not shown).
Figure 1. pADPr Is Enriched in SGs upon Multiple Types ofStresses and Modifies Specific Cytoplasmic RNA-Binding Proteins Dependent on
(A) pADPr staining using LP96-10 antibodies in HeLa cells untreated or treated with 100 mM arsenite for 60 min or for 30 min followed by 100 mM arsenite +
100 mg/ml cycloheximide for 30 min. Arrowheads, SGs; scale bar, 10 mm.
(B) HeLa cells treated with 100 mM arsenite were stained for pADPr, SG component Ago2 (arrowheads), or PB component GE-1 (arrows). DNA was stained with
Hoechst 33342 (blue); scale bar, 10 mm.
(C) Immunoprecipitates of four GFP-tagged SG-localized RNA-binding proteins from cells treated with or without 20 nM pateamine A were probed for pADPr.
(D) Immunoprecipitates of GFP-Ago2 from cells treated with or without 20 nM pateamine A were probed for pADPr. The cell extracts either included or excluded
1 mM ADP-HPD and with or without 1 mM NAD+before immunoprecipitation by anti-GFP.
(E) pADPr modification of Ago2 from cells treated with or without 20 nM pateamine A was verified by treating the immunoprecipitates with ARH3. The immu-
noprecipitates were probed for pADPr (left) and GFP (right).
(F) Immunoprecipitates of wild-type and PIWI mutant of GFP-Ago2 from cells treated with or without 20 nM pateamine A were probed for pADPr. For (C)–(F), cell
extracts included 1 mM ADP-HPD unless stated otherwise; shown are western blots for pADPr (LP96-10) and GFP levels in each immunoprecipitate. Asterisks
indicate the position of the corresponding GFP-tagged RNA-binding protein constructs. Black dots indicate nonspecific binding to BSA by LP96-10. See also
RNA Regulation by Cytoplasmic PARPs
490 Molecular Cell 42, 489–499, May 20, 2011 ª2011 Elsevier Inc.
stress conditions that result in SG assembly, including heat
shock, glucose deprivation, treatment with the proteasome
inhibitor MG132, or with translation initiation inhibitors (pate-
amine A and hippuristanol) (Figure S1C and data not shown).
Together, these data indicate that pADPr is enriched in SGs
upon multiple stresses and contain polymers of at least 20
SGs can be disassembled by cycloheximide, which shifts the
equilibrium of mRNA pools from stalled initiation complexes at
SGs to polyribosomes (Anderson and Kedersha, 2008). Upon
addition of cycloheximide to arsenite-treated cells, pADPr no
longer localized as punctate structures (Figure 1A, rightmost
panel), suggesting that the pADPr originally at SGs relocated
with mRNA and/or their binding proteins. Thus, pADPr colocali-
zation with other mRNA-binding proteins was further analyzed
(Anderson and Kedersha, 2008). pADPr colocalized with
miRNA-binding protein Ago2, RNA decay factor G3BP1, transla-
tional suppressor TIA-1, and poly(A)-binding protein PABP
(Figures 1B and S1D). In contrast, no significant pADPr colocal-
ization was observed with GE-1, a marker for P-bodies, another
cytoplasmic structure enriched in RNA-binding proteins (Figures
1B and S1E and legends for statistics). Thus, pADPr localization
to SGs is not the result of nonspecific binding of pADPr with
pADPr Modification of Cytoplasmic RNA-Binding
Proteins Requires the Presence of an RNA-Binding
pADPr also modifies other targets (Schreiber et al., 2006).
Because Ago2, G3BP1, TIA-1, and PABP colocalized with
pADPr at SGs, we examined their pADPr modification status.
Proteins were expressed as GFP-fusions, immunoprecipitated
from extracts of either unstressed or stressed cells in the pres-
ence of PARG-specific inhibitor ADP-HPD, then immunoblotted
for pADPr. If the protein is modified, a slower migrating material
stainedpositively withanti-pADPr isexpected and wouldappear
as a ‘‘smear’’ as a result of heterogeneity in the length of polymer
attached (Figure 1C). Such pADPr-positive material was
observed for Ago2, G3BP1, and TIA-1, but not PABP. Upon
stress, Ago2, G3BP1, and TIA-1, but not PABP, exhibited
significantly increased pADPr modification (Figure 1C). Similarly,
other Argonaute family members—Ago1, Ago3, and Ago4—are
modified by pADPr during nonstress conditions, suggesting
that all Argonaute proteins are actively regulated by pADPr in
unstressed cells and such modification increases upon stress
The presence of pADPr modification was further verified for
Ago2 in four different ways. First, endogenous Ago2 was immu-
noprecipitated from unstressed and stressed cells and probed
for pADPr (Figure S1G). Similar to GFP-Ago2, endogenous
Ago2 exhibited moderate amounts of pADPr modification during
nonstress conditions and the modification increased upon
stress. Second, the pADPr staining associated with GFP-Ago2
under nonstress and stress conditions decreased when PARG-
specific inhibitor ADP-HPD was omitted, and increased upon
addition of NAD+, a substrate for ADPr synthesis, suggesting
that pADPr modification of Ago2 is actively regulated even in
in vitro conditions (Figure 1D). Third, treatment of a pADPr glyco-
hydrolase, ARH3, but not RNase A, eliminated the anti-pADPr
signal from the immunoprecipitates (Figure 1E and data not
shown). These slow-migrating, pADPr-positive materials were
also GFP-positive, confirming that the observed signal was
activities in the GFP-Ago2 immunoprecipitates were further
examined by incubating with increasing concentrations of
NAD+containing trace amounts of radioactive NAD+. The reac-
tions were then resolved via SDS-PAGE and were visualized
by autoradiography (Figure S1H). A heterogeneous mobility of
radioactive signal was observed immediately above where
GFP-Ago2 migrated (denoted by asterisk). The intensity of this
signal was directly dependent on the concentration of NAD+
added. These data suggest that PARPs associated with Ago2
in the immunoprecipitates can synthesize pADPr. Importantly,
addition of the general PARP inhibitor 3-aminobenzamide (3-
AB) to the reactions completely eliminated any incorporation of
radioactivity in the Ago2 precipitates, suggesting that the signal
observed is due to ADP-ribosylating activities (Figure S1H).
Because pADPr modification was observed onmRNA-binding
proteins Ago2, G3BP1, and TIA-1, we next tested whether their
modification requires mRNA binding. To test this, fragments of
each protein were expressed at levels comparable to wild-
type, immunoprecipitated from unstressed and stressed cells,
and immunoblotted for pADPr. In all cases, pADPr modification
required the presence of an mRNA-binding domain, such as
Ago2’s PIWI domain (Figure 1F) or RRM in G3BP1 and TIA-1
(Figures S1I and S1J) in both nonstress and stress conditions.
These data suggest that either the pADPr modification site is
within or proximal to theRNA-binding domains of theseproteins,
is/are associated via mRNAs.
Specific PARPs and PARG Isoforms Associate
with Cytoplasmic mRNP Complexes
We reasoned that PARPs responsible for such modification are
likely associated with these cytoplasmic RNA-binding proteins
during nonstress conditions and that these associations may
be retained in SGs upon stress. Thus, SGs were used as a surro-
gate to identify such PARPs. We screened a library of GFP
fusions (S.V., unpublished data) to 17 of the 18 human PARPs
(PARP-13.2 isoform included, but not PARP-14 because the
full-length cDNA was unavailable) for colocalization with known
SG markers (Figures 2A and S2A and data not shown). Five of
17 PARPs localized to SGs: PARP-5a, -12, -13.1, -13.2, and
-15 (Figure S2B). Specific SG localization of these PARPs was
confirmed using live-cell imaging. In each case, GFP-tagged
PARPs strongly colocalized with RFP-G3BP1 at the assembling
SGs (Movie S1). To further rule out overexpression artifacts, we
repeated the colocalization screen in HeLa (Figure 2B) and
RPE-1 cells (Figure S2C) using a library of affinity purified and
characterized antibodies against each PARP (S.V., unpublished
data).Antibody stainingconfirmed theGFP-PARP screenresults
and also identified PARP-14 as a SG protein (confirmed via two
distinct antibodies) (Figures 2B and S2C and data not shown).
Consistent with the GFP localization screen, the remaining
RNA Regulation by Cytoplasmic PARPs
Molecular Cell 42, 489–499, May 20, 2011 ª2011 Elsevier Inc. 491
12 PARPs did not localize to SGs endogenously, confirming
these six PARPs as SG proteins (hereafter referred as SG-
PARPs) (Figure S2B).
We next examined the localization of pADPr-hydrolyzing
enzyme PARG. Immunostaining suggests that a fraction of
endogenous PARG localizes to the cytoplasm (Figure 2B).
Similar to the SG-PARPs, PARG was enriched in SGs upon
stress (Figure 2B). Three major PARG isoforms exist: PARG99,
PARG102, and PARG110 (Figure S2D). To determine which
localize to SGs, GFP fusions of each were screened. Both
PARG99 and PARG102 strongly colocalized with SGs upon
stress and were dispersed in the cytoplasm during nonstress
Figure 2. Specific PARPsandPARGIsoformsLocalizeintheCytoplasmicSGsandtheLevelofPARGModulatestheKineticsofSGAssembly
(A) Summary of SG localization screen of PARP family. Green shading indicates SG-PARPs as determined by GFP-PARP fusions or PARP-specific antibodies.
(B) HeLa cells were treated with or without 100 mM arsenite for 60 min and stained using antibodies against SG-PARPs and PARG.
(C) HeLa cells expressing GFP-tagged PARG isoforms were treated with or without 250 mM arsenite for 30 min.
(D) Overexpression of cytoplasmic PARG isoforms inhibits SG assembly. Experiment performed as in (C), but heat map shows level of GFP-PARG isoforms (99,
102, 110) compared with untransfected control (C). Accompanying graph shows quantitation of image data (R200 cells for each condition from at least six
independent fields). Cells with GFP intensity above background are classified as ‘‘High’’ whereas cells with intensity indistinguishable from background levels as
‘‘Low/Undetected.’’ Paired t test p < 0.01 (**), derived from comparison to untransfected control; error bars indicates SD.
(E) pADPr hydrolysis is required for SG disassembly. Shown are representative images taken 0,30, and 60 min after washout of 30 min 100 mMarsenite treatment
in control and PARG knockdown HeLa cells. Quantitation: R100 cells for each condition, n = 3. Paired t test p < 0.01 (**) and error bars indicate SD.
anti-eIF3, and DNA by Hoechst 33342 (blue); scale bars = 10 mm. See also Figure S2 and Movie S1.
RNA Regulation by Cytoplasmic PARPs
492 Molecular Cell 42, 489–499, May 20, 2011 ª2011 Elsevier Inc.
conditions. PARG110 was nuclear and unaffected by stress
(Figure 2C). Although nuclear PARG110 is known to function in
DNA damage repair (Schreiber et al., 2006), PARG99 and
PARG102 isoforms are functionally uncharacterized; thus, their
identification at SGs uncovers potential function for these cyto-
The presence of six PARPs and two PARG isoforms in SGs
suggests that the pADPr concentration may be dynamically
regulated there. Overexpression of each SG-PARP resulted in
denovo assembly of SGs without altering eIF2aphosphorylation
levels (Figure S2E). This finding suggests that these enzymes
and/or their product, pADPr, play a structural role in SG function
rather than causing a general impairment of translation via eIF2a
inhibited SG assembly, whereas overexpression of nuclear
PARG110 had no effect (Figure 2D). Moreover, three distinct
siRNAs targeting the first exon of PARG102 coding region
individually delayed SG disassembly (Figures 2E and S2F). No
such effect was observed upon knockdown of a nuclear PARG
ARH3 (Figure S2G). Interestingly, all tested proteins that can
nucleate SGs upon overexpression, including Ago2, TIA-1, and
G3BP1, but not nonnucleator PABP (Anderson and Kedersha,
2008), serve as acceptors of pADPr modification (Figure 1C),
suggesting a strong correlation between polymer synthesis
and SG assembly. Consistent with this, no pADPr modification
was identified on mutants of TIA-1 and G3BP1 that are dominant
negative in SG formation (Kedersha et al., 1999; Tourriere et al.,
2003) (Figures S1I and S1J). Taken together, these data suggest
that both assembly and maintenance of SG structure depends
on regulating pADPr concentration locally in the cytoplasm.
Stress or Specific PARP Overexpression Alleviates
To identify a possible function for pADPr modification of RNA-
binding proteins on their activities, we focused on Argonautes
dent on miRNAs (Leung et al., 2006) and pADPr modification of
Ago2 increases upon stress,we examined miRNA activity during
stress. miRNA activity was monitored using a luciferase reporter
that contains six bulged sites for an siRNA, siCXCR4, in its 30
UTR (Doench et al., 2003). The luciferase protein contains
a destabilization signal that reduces its half-life to 20 min so
that any change in expression is rapidly monitored. Under
nonstress conditions, the luciferase construct was repressed
15-foldbytargetingsiCXCR4 relativeto acontrol siRNA.Consis-
tent with Bhattacharyya et al. (2006), we observed a relief of
miRNA-mediated silencing under stress conditions. Under
stress conditions, the relative fold repression is reduced to ?5-
rate was globally reduced; however, the expression of the lucif-
erase mRNA targeted by siCXCR4 decreased to a lesser extent
than the nontargeted reporter.
in the absence of exogenous stress, we next examined whether
miRNA activity is correlated with SG induction or specific
SG-PARP overexpression (Figure 3B). Interestingly, overexpres-
sion of PARP-13.1 or PARP-13.2 each resulted in a ?3-fold
decrease in repression of miRNA activity, and overexpression
of PARP-12 resulted in a modest ?1.8-fold decrease, whereas
overexpression of other SG-PARPs or another SG nucleator
G3BP1 had no effect. As controls, overexpression of GFP had
no effect, whereas the repression can be relieved by a competi-
tive inhibitor miRNA sponge (Ebert et al., 2007). Thus, the
observed decrease in miRNA-mediated repression is not likely
due to an overall increase in SG assembly or SG-PARP overex-
pression, but instead is due to the specific function of PARP-
To further explore the role of PARP-13 in miRNA silencing, the
association between PARP-13 and Ago2 was investigated (Fig-
ure 3C). Immunoprecipitation of endogenous Ago2 identified
Figure 3. Stress or PARP-13 Overexpression Alleviates miRNA-
(A) miRNA activity assay in untreated 293T cells or cells treated with 30 nM
pateamine A (PatA), 1 mM hippuristanol (Hipp), or 250 mM arsenite (As) for 2 hr,
panel) in the presence of the targeting siRNA normalized to a control siRNA;
n = 3.
(B) miRNA activity assay upon overexpression of SG-PARPs. Relative fold
paired t test p < 0.05 (*) and < 0.01 (**).
(C) Antibodies against endogenous Ago2 were used for immunoprecipitation
from cytoplasmic extract of HeLa cells treated with or without 250 mM arsenite
for 30 min. On the right, the extract was pretreated with 200 mg/ml RNase A for
20 min at 25?C.
(D) Immunoprecipitates of GFP-tagged PARP-13.1 or -13.2 from cells treated
with or without 20 nM pateamine A for 30 min were probed for pADPr, where
cell extracts included 1 mM ADP-HPD. Asterisks indicate the position of the
corresponding GFP-tagged PARPs. See also Figure S3.
RNA Regulation by Cytoplasmic PARPs
Molecular Cell 42, 489–499, May 20, 2011 ª2011 Elsevier Inc. 493
PARP-13.1 and PARP-13.2 as binding partners mediated by
mRNA. Given that the amount of PARP-13 bound to Ago2
remains unchanged in nonstress and stress conditions, it seems
that other properties, such as posttranslational modifications, of
PARP-13 are likely altered during stress, resulting in enhanced
We therefore examined whether PARP-13 proteins are modi-
fied by pADPr. GFP-PARP-13.1 and -PARP-13.2 were immuno-
precipitated from unstressed or stressed cells and probed for
pADPr. Both isoforms were modified under nonstress condi-
tions. Remarkably, PARP-13.2 exhibited a dramatic increase in
modification upon stress, whereas PARP-13.1 modification
remained roughly the same (Figure 3D). Similarly, immunopre-
cipitation of endogenous PARP-13 via a pan-PARP-13 antibody
demonstrated an increase in pADPr modification upon stress
(Figure S3A). PARP-13 modification was further confirmed by
antibody 10H, which recognizes pADPr with R 20 subunits
by glycohydrolase ARH3 or PARG (Figure S3C) but not RNase A
(data not shown). Interestingly, the amount of pADPr modifica-
tion shifted among the SG-PARPs during stress conditions;
similar to PARP-13.1, there was no change in PARP-12 modifi-
cation, whereas PARP-5a and PARP-15 modifications were
reduced (Figure S3D). These results suggest a possible shift in
PARP activity from auto-modification to modification of stress
targets, including Ago1-4, TIA-1, G3BP1, and PARP-13 family
PARP-13 Family Members Are pADPr-Modified
by Other SG-PARPs
The pADPr modifications attached to PARP-13 proteins are not
due to auto-ADP-ribosylating activities because PARP-13.1’s
PARP domain is inactive in vitro and PARP-13.2 lacks
a PARP domain (Kleine et al., 2008). To determine which
PARPs modify PARP-13, we examined the catalytic activity of
each SG-PARP. GFP-tagged SG-PARPs were purified by
immunoprecipitation from either unstressed or stressed cells
(PARP-1 serves as a positive control), and the immunoprecipi-
tates were washed twice with buffer containing high salt
(450 mM NaCl) and then divided into equal aliquots for ADP-
ribosylating activity (Figures 4A and S4A). As expected,
PARP-1 and PARP-5a demonstrated auto-poly(ADP-ribosylat-
ing) activities, as shown by the mobility shifts above their
respective molecular weights (asterisks in Figure 4A). On the
other hand, PARP-12 and PARP-15 exhibited mono(ADP-ribo-
sylating) activities, as demonstrated by single radioactive
bands at their respective molecular weights. Consistent with
their lack of active PARP domains, significant amounts of radio-
activity were not associated with PARP-13.1 or PARP-13.2.
Interestingly, PARP-12 and PARP-15 samples also contained
single bands of radioactivity near the expected mobility of
endogenous PARP-13.1 (circle) and PARP-13.2 (triangle).
Similar results were observed from stress conditions (data not
shown). These results suggest that PARP-12 and PARP-15
could be part of complexes that modify PARP-13 family
members even in high salt conditions.
To determine whether SG-PARPs bind to and modify one
another, the binding interactions between different PARPs
were analyzed in the immunoprecipitates. Samples were
analyzedeither byimmunoblot(Figure 4B)or massspectrometry
(LC-MS/MS) (Figure S4B). Both endogenous PARP-13 isoforms
were detected in immunoprecipitates from PARP-12, -13.1,
-13.2, and -15 by both methods. The interaction between
PARP-13.1 and PARP-13.2 confirms that both isoforms function
as a complex (Law et al., 2010). In comparison, the associations
of PARP-5a and both PARP-13 isoforms are relatively weak,
because they were only detected by immunoblot but not by
mass spectrometry. However, the association with PARP-13 is
specific with these PARPs because it was not observed with
the negative control, GFP (Figure 4B). Interestingly, we also
observed an association between PARP-12 and PARP-5a.
Figure 4. PARP-13 Family Members Are Poly(ADP-Ribosylated) by
(A) In vitro ADP-ribosylating assay for PARP-1, -5a, -12, -13.1, -13.2, and -15.
HeLa S3 cells were transfected with individual GFP-tagged SG-PARPs, and
GFP-PARP immunoprecipitates were washed twice with 450 mM NaCl, then
once with 150 mM NaCl. The immunoprecipitates were incubated with 0, 25,
50, 100, or 200 mM NAD+(with 1/175-fold of P32-labeled NAD+) at 16?C for
Asterisks indicate the position of the corresponding GFP-tagged SG-PARPs;
(B) Western blots of the immunoprecipitates from (A) were probed with PARP-
5a, -12, and -13 (antibodies for PARP-15 are not good for detecting endoge-
nous protein). Immunoprecipitates from cells transfected with GFP were used
as a negative control.
(C) HeLa S3 cells were transfected with GFP-tagged Ago2 and cells either
treated with (+) or without (?) 20 nM pateamine A for 30 min. Untransfected
cells treated with 20 nM pateamine A were used as a negative control (ø). The
cytoplasmic lysates were immunoprecipitated with anti-GFP and washed
thrice with cytoplasmic lysis buffer. The input and immunoprecipitates were
probed with antibodies against PARP-1, -5a, -12, and -13. See also Figure S4.
RNA Regulation by Cytoplasmic PARPs
494 Molecular Cell 42, 489–499, May 20, 2011 ª2011 Elsevier Inc.
Therefore, it seems likely that individual PARPs associate with
other PARPs and account for their respective ADP-ribosylating
activities in vitro and in cells.
In light of these findings, we re-examined whether Ago2 binds
to other SG-PARPs. GFP-Ago2 was immunoprecipitated from
either unstressed or stressed cells and probed for individual
between GFP-Ago2 and PARP-5a and PARP-12, in addition to
PARP-13, in both unstressed and stressed cells. No such asso-
ciation was observed for the nuclear PARP-1. Such association
of Ago2 with catalytically active PARPs is consistent with the
in vitro ADP-ribosylating activities observed in GFP-Ago2 immu-
noprecipitates (Figure S1H).
Knockdown of Glycohydrolase PARG Alleviates
Because pADPr modification is dynamically regulated by PARP
and PARG activities, we next examined the effect of miRNA
activity upon knocking down PARG. PARG knockdown results
in an increase in pADPr modification on endogenous Ago2 (Fig-
ure 5A) and a ?5-fold decrease in miRNA-mediated repression
(Figures 5B and S5A). Thus, similar to stress conditions, an
Figure 5. PARG Knockdown Alleviates miRNA-Mediated Repression and miRNA-Directed Cleavage
(A) pADPr modification levels of endogenous Ago2 in HeLa S3 cells transfected with 25 nM control siRNA or siPARG for 48 hr. Asterisk indicates where Ago2
migrated. Shown are western blots for Ago2, PARG, and tubulin.
(B) 293T cells were transfected with 25 nM control siRNA or siPARG for 72 hr. Relative fold repression was measured as in Figure 3A; n = 3.
(C)PARG knockdown effectobservedinluciferase reporter withseven artificial miR-20binding sites. Therelative fold repression wascalculated bytheamountof
expression oftheconstructnormalized toaconstructwithallbindingsitesmutatedattheirseedpositions. Theassaywastested withexogenous additionofmiR-
20 (left) or with endogenous miR-20 (right); n = 4.
(D) PARG knockdown effect observed in luciferase reporter with endogenous HMGA2 30UTR. The relative fold repression is calculated by the amount of
expression by the wild-type construct (Luc-wt) normalized to the mutant construct Luc-m7; n = 4.
to show cells transfected with a control siRNA.
(F) The effect of PARG knockdown on miRNA-directed cleavage wasexamined for luciferase construct withone perfect siCXCR4-,let-7-, or miR-20-binding site;
n = 4 in each case.
(G) The effect of SG-PARP overexpression on miRNA-directed cleavage assay as in (F); n = 5.
(H) The effect of stress on miRNA-directed cleavage was tested with a luciferase reporter with two perfect binding sites for siCXCR4 using the same transfection
conditions and drug treatment as in (E); n = 3. For (B)–(H), error bars indicate SD; paired t test p < 0.05 (*) and < 0.01 (**). See also Figure S5.
RNA Regulation by Cytoplasmic PARPs
Molecular Cell 42, 489–499, May 20, 2011 ª2011 Elsevier Inc. 495
inverse correlation between the pADPr modification of Ago2 and
miRNA-mediated repression was observed upon PARG knock-
down in nonstress conditions.
conditions was further examined with other constructs. Using
a luciferase construct with seven miR-20 binding sites, the
expression was reduced ?15-fold upon addition of exogenous
miR-20, but such repression was reduced by half upon PARG
knockdown (Figure 5C). Given that miR-20 is expressed in
293T cells (Landgraf et al., 2007), the repression mediated by
endogenous miR-20 was examined. Under this condition, ?11-
fold repressionwas observed, and suchrepression wasreduced
1.8-fold in siPARG-transfected cells (Figure 5C). Similarly,
miRNA-mediated repression was examined with a luciferase
construct (Luc-wt) fused with the endogenous HMGA2 30UTR,
which contains seven let-7-binding sites (Mayr et al., 2007)
(Figure 5D). As a control, a luciferase (Luc-m7) construct with
each miRNA-binding site mutated was used to normalize
expression. Upon addition of exogenous let-7, the wild-type
construct was repressed by 4.5-fold compared to the mutant.
On the other hand, there is no such repression upon addition
of miR-631, which does not bind anywhere in the HMGA2 30
UTR. Similar to other constructs, the let-7-mediated repression
is reduced by half upon PARG knockdown.
Because both PARG knockdown and stress can alleviate
miRNA-mediated silencing individually, we asked whether the
combination of both results in further relief in miRNA-mediated
silencing (Figures 5E and S5B). Indeed, a significant further
reduction of miRNA-mediated repression was observed upon
stress in siPARG-treated cells, though the magnitude is less
than multiplicative. Although stress resulted in a ?3.4-fold
repression in control cells, only ?1.6-fold repression was
observed upon stress in siPARG-treated cells. Given that stress
and PARG knockdown did not synergistically attenuate miRNA
silencing, it is likely that the stress pathway involved in modu-
lating the miRNA-mediated silencing is not independent of the
pathway that regulates pADPr level via PARG.
PARG Knockdown, PARP-13 Overexpression, or Stress
Alleviates miRNA-Directed Cleavage
Apart from inhibiting translation or accelerating mRNA decay,
miRNAs can also induce mRNA cleavage when the miRNA binds
to its mRNA target in a perfectly complementary manner (e.g.,
Yekta et al., 2004). The effect of PARG knockdown on the
miRNA-directed cleavage was next examined. First, a luciferase
construct with a perfectly complementary binding site for
of siCXCR4 or let-7, expression is reduced 15- or 7-fold, respec-
tively. As in the case of the constructs with bulged configuration
for siRNA/miRNA, this repression is relieved upon PARG knock-
down (4.5-fold for siCXCR4 and 2-fold for let-7). Such relief in
repression upon PARG knockdown was also examined for
?1.2-fold upon PARG knockdown (Figure 5F). Because Ago2
is the only member of the Argonaute family that is capable of
mediating miRNA-directed cleavage, these statistically signifi-
cant decreases in repression must reflect inhibition of a complex
containing this factor.
Given that the relief of miRNA-mediated translational inhibi-
tion/mRNA decay can result from overexpression of specific
PARPs or stress, their effects on miRNA-directed cleavage
were also examined. In the reporter construct containing one
(or two) perfect siCXCR4-binding site(s), overexpression of
PARP-13.1 and -13.2 reduced the level of miRNA-mediated
directed cleavage by 1.3- (1.6-) and 1.4- (1.7-) fold, respectively
(Figures 5G and S5D). Thus, both major miRNA-mediated
processes involve PARP-13 family members. Similarly, stress
reduced the repression ?2.5-fold using the reporter containing
two perfect siCXCR4-binding sites (Figures 5H and S5E).
When stress and siPARG treatment were combined, the repres-
sion was reduced to ?1.6-fold (Figures 5H and S5F). Thus,
similar to miRNA-mediated silencing (Figure 5E), the reduced
magnitude observed upon stress in siPARG-treated cells, as
compared to untreated cells, is likely because the stress
pathway involved in modulating the miRNA-directed cleavage
overlaps with the pathway that regulates pADPr level via PARG.
pADPr Regulates Posttranscriptional Gene Expression
in the Cytoplasm
We report a previously uncharacterized function for pADPr—
cytoplasmic posttranscriptional regulation of mRNA. pADPr
regulates miRNA silencing and catalyzes the assembly of micro-
scopically visible SG structures. This posttranscriptional regula-
tion occurs in the interphase cytoplasm, further extending the
function of pADPr outside the nucleus. Our findings help explain
several intriguing observations regarding pADPr function in the
cytosol. First, pADPr synthesis and hydrolysis activities are
enriched in postnuclear, postmitochondrial fractions and in the
free mRNP fractions (Elkaim et al., 1983; Thomassin et al.,
1985). Free mRNP fractions are enriched in factors that regulate
translation and decay of mRNAs, many of which are SG com-
ponents. Second, our data indicate potential functions for the
two cytoplasmic isoforms of PARG, which together are more
active than the single nuclear isoform (Meyer-Ficca et al.,
2004). Third, large amounts of RNA-binding proteins were asso-
ciated with pADPrin ourprevious analysesandbyglobalproteo-
mic analyses; some of them are SG components, including
G3BP1 and PABP (Chang et al., 2009; Gagne et al., 2008).
Here, we report that specific cytoplasmic RNA-binding
proteins—Ago2, G3BP1, and TIA-1—are pADPr-modified de-
pending on the presence of their RNA-binding domains. Each
protein is increasingly modified by pADPr upon stress and en-
riched in SGs (though not all components, such as PABP, are
modified). Perhaps one general function of pADPr is to recruit
RNA-binding proteins to specific locations, such as SGs, thus
functioning as a scaffold for protein recruitment. This scaffold
plays in other complexes or structures, such as the mitotic
spindle, where pADPr recruits spindle pole proteins (Chang
et al., 2005, 2009); Cajal bodies, a nuclear organelle enriched in
nucleic acid-binding proteins that can bind pADPr (Kotova
et al., 2009); or at DNA damage sites, pADPr recruits nucleic
acid-binding proteins for chromatin remodeling and DNA repair
(Ahel et al., 2009). However, in contrast to these examples
RNA Regulation by Cytoplasmic PARPs
496 Molecular Cell 42, 489–499, May 20, 2011 ª2011 Elsevier Inc.
involving the activation of a single PARP, we identified multiple
PARPs in SGs from distinct subfamilies: Tankyrase PARP-5a;
RNA-binding PARP-12 and PARP-13 isoforms; and PARP-14
and PARP-15 that contain pADPr-binding macro-domains.
These pADPr-synthesizing activities along with PARG99 and
PARG102 isoforms likely regulate the local pADPr concentration
that determines the assembly and maintenance of SGs.
pADPr Regulates miRNA Function in the Interphase
miRNA targets are preferentially expressed relative to total
protein synthesis under three conditions: stress, PARP-13 over-
expression, and PARG knockdown. Two of these conditions,
PARP-13 overexpression and stress, trigger SG assembly. Yet
this apparent correlation is paradoxical because SGs are not
sites of active translation as they do not contain 60S ribosomes
(Anderson and Kedersha, 2008). Therefore, any preferential
translation of miRNA targets probably occurs outside SGs in
the cytoplasm. Several results suggest that the two phenomena
are likely to be coincidental events that are not necessarily
mechanistically linked. For example, overexpression of G3BP1,
a known inducer of SG assembly, does not result in the relief
of miRNA silencing, whereas PARG knockdown results in the
relief of miRNA silencing in the presence or absence of SG
formation. This is not surprising because the majority of Ago/
miRNA complexes are located in the diffuse cytoplasm; only
5% are localized in SGs upon stress and such pool is rapidly
exchanging with the cytoplasm (Leung et al., 2006). Thus, the
relief of miRNA silencing as a result of poly(ADP-ribosylation)
likely occurs in the diffuse cytoplasm.
At what step does pADPr modulate miRNA silencing? Given
that the miRNA silencing is alleviated upon increase in pADPr
modification level for both endogenous miRNAs and exoge-
nously added siRNA, pADPr likely regulates a step downstream
of miRNA processing in the cytoplasm. Consistent with this, the
expression levels of nearly all (>99%) miRNAs examined using
miRNA microarray remained unchanged upon PARG knock-
down (data not shown). In addition, the relief of miRNA silencing
was observed in constructs that can be cleaved through
perfectly complementary sites and silenced through partially
complementary sites. Thus, pADPr likely regulates miRNA func-
tion at a step upstream of the direct activity of the Argonaute
Here, we propose that the accessibility of Argonaute/miRNA
complex to its target mRNA is affected by an increase in local
pADPr modification on multiple proteins that bind to the target,
resulting in the relief of miRNA silencing (Figure 6). Those
proteins that are modified by pADPr include all Argonaute
members and PARP-5a, -12, -13.1, and -13.2, among which
Ago1-4 and PARP-13.2 are increasingly modified upon stress
when miRNA silencing is relieved. Consistent with the impor-
tance of poly(ADP-ribosylation), relief of miRNA silencing was
observed upon PARG knockdown when pADPr modification
on proteins is generally increased. Such high concentration of
negatively charged pADPr modification near the sites of
miRNA:mRNA-binding likely disrupts the electrostatic interac-
tion between similarly charged miRNA and mRNA. Alternatively,
the sizeable pADPr modification might cause steric hindrance to
prohibit effective miRNA silencing. Recently, it has been shown
that in vitro addition of pADPr inhibits the RNA-binding ability
of a Drosophila heterogeneous nuclear ribonucleoprotein (Ji and
Tulin, 2009). Given that pADPr also exhibited binding affinity to
RNA-binding proteins (Chang et al., 2009; Gagne et al., 2008),
one function of pADPr could be to regulate the binding of
RNAs to RNA-binding proteins.
One interesting observation from this study is that overexpres-
sion of PARP-13 family members affects both miRNA-mediated
repression and miRNA-directed cleavage, yet these PARPs are
not catalytically active. Instead, our data suggest that PARP-5a,
PARP-12, and PARP-15 are likely the source of pADPr
poly(ADP-ribosylating) activities and PARP-12 and PARP-15
mono(ADP-ribosylating) activities, (2) PARP-5a and PARP-12,
but not PARP-1, associate with Ago2, and (3) all of these PARPs
associate with endogenous PARP-13 family members. We note
that the association of an inactive PARP with active PARPs
though itself has no active kinase domain, can mediate signaling
through heterodimerization with active EGF family kinases like
Her2 (Holbro et al., 2003). Perhaps, because of their ability to
bind mRNA, the function of the inactive PARP-13 isoforms is to
anchor the activity of the catalytically active PARPs to the
domain is required for pADPr modification of Ago2.
In conclusion, our data point to two functions for pADPr in the
cytoplasm. At SGs, pADPr modulates the assembly and mainte-
nance of an mRNP-enriched structure. At submicroscopic
miRNP complexes, pADPr relieves miRNA-mediated repression
and miRNA-directed cleavage under stress conditions. These
cytoplasmic functions are likely mediated through the concerted
activities of catalytically inactive and mADPr- and pADPr-
synthesizing PARPs. Such cross-subfamily mechanism of
pADPr synthesis suggests that pADPr polymerization could be
Figure 6. A Working Model: A High Local Concentration of pADPr at
miRNA Complex Results in Relief of miRNA Silencing
Upon stress, multiple proteins including all Argonaute family members and
PARP-13.1/2 complex are increasingly modified by pADPr. Such increase in
poly(ADP-ribosylation) during stress could be due to increase in PARP activity
and/or decrease in PARG activity (dotted line). High concentration of pADPr
near the Argonaute/miRNA complex might disrupt electrostatic interaction or
cause steric hindrance for effective miRNA silencing. Similar relief of miRNA
silencing is also observed upon overexpression of PARP-13 or, conversely,
upon knockdown of PARG.
RNA Regulation by Cytoplasmic PARPs
Molecular Cell 42, 489–499, May 20, 2011 ª2011 Elsevier Inc. 497
more complex than previously thought. Given that other post-
transcriptionalfactors suchas G3BP1and TIA-1 areincreasingly
modified by pADPr upon stress and that these modifications
depend on the presence of an RNA-binding domain, it is likely
Following SG induction, coverslips were rinsed twice in PBS and either
extracted for 30 s with buffer A, fixed for 15 min in 4% paraformaldehyde in
buffer B, or fixed in ice-cold methanol for 5 min, followed by slow rehydration
with PBS. Coverslips were incubated with 1?antibodies for 45 min and 2?anti-
bodies for 35 min. See Supplemental Experimental Procedures for buffer
HeLa S3 cells (8 3 107) were transfected with GFP-PARPs or RNA-binding
proteins for 48 hr using 293fectin, such that the expression did not induce
visible SGs. Cells were either treated with or without 20 nM pateamine A or
250 mM Arsenite for 30 min. At 10 min before stress, latruculin B was added
to 0.5 mg/ml. Cells were lysed with cytoplasmic lysis buffer C. Lysate was
spun at 18,407 g for 10 min and a final concentration of 10 mg/ml cytochalasin
B and 25 mM nocodazole was added to the supernatant. The supernatants
cipitates were washed for 10 min with buffer C, then twice in buffer C contain-
ing 300 mM NaCl and again with buffer C. Beads were then eluted with sample
buffer and heated for 10min at70?C. For endogenous Ago2 modification, anti-
Ago2 antibody was preincubated with Protein A beads. The supernatant was
made by spinning the lysate at 2,300 g or 18,407 g for 10 min, and the beads
were washed 3 3 5 min with buffer C before eluting with sample buffer. See
Supplemental Experimental Procedures for buffer receipes.
miRNA Reporter Assay
Twenty-four hours after transfection, cells were either untreated or stressed
with 30 nM pateamine A, 1 mM hippuristanol, or 250 mM arsenite for 2 hr before
lysis for luciferase assay. Firefly and Renilla luciferase signals were measured
using the Dual Luciferase reporter assay system (Promega). Shown are repre-
sentative luciferase assay results from 3–5 replicates, as indicated in the figure
legend,ineach experimental condition, and each condition hasbeen indepen-
dently examined 2–4 times.
Supplemental Information includes Supplemental Experimental Procedures,
five figures, and one movie and can be found with this article online at
We thank M. Miwa, J. Tazi, P. Anderson, N. Kedersha, and J. Pelletier for kind
gifts of reagents; A. West, T. Lai, and E. Vasile for technical support; A. Young,
M. Ebert, J. Wilusz, and P. Boutz for comments; and M. Lindstrom for illustra-
tions.A.K.L.LwasaspecialfellowofLeukemia andLymphomaSociety. P.C.is
a Rita Allen Foundation Scholar, a Kimmel Foundation for Cancer Research
Scholar, and a Howard S. and Linda B. Stern Career Development Professor.
This work was supported by the National Institutes of Health (grant RO1-
CA133404 to P.A.S.), the National Cancer Institute (grant PO1-CA42063 to
P.A.S.), and partially by Cancer Center Support (core; grant P30-CA14051).
Received: December 8, 2010
Revised: March 11, 2011
Accepted: April 25, 2011
Published: May 19, 2011
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