text:
Page 1
MOLECULAR AND CELLULAR BIOLOGY, Nov. 2011, p. 4417–4429
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 31, No. 22
A Novel Role for hSMG-1 in Stress Granule Formation?
James A. L. Brown,1,2† Tara L. Roberts,1,3† Renee Richards,1,3Rick Woods,1Geoff Birrell,1
Y. C. Lim,1,3Shigeo Ohno,4Akio Yamashita,4Robert T. Abraham,5
Nuri Gueven,1and Martin F. Lavin1,3*
Radiation Biology and Oncology Laboratory, Queensland Institute of Medical Research, Brisbane, Queensland 4029, Australia1;
Chromosome Biology Centre, National University of Ireland, Galway, Ireland2; The University of Queensland Centre for
Clinical Research, Brisbane, Queensland 4029, Australia3; Department of Molecular Biology,
Yokohama City University School of Medicine, Yokohama 236-0004, Japan4; and
Wyeth Oncology Research, 401 N. Middletown Road, Pearl River, New York 109655
Received 27 July 2011/Returned for modification 25 August 2011/Accepted 29 August 2011
hSMG-1 is a member of the phosphoinositide 3 kinase-like kinase (PIKK) family with established roles in
nonsense-mediated decay (NMD) of mRNA containing premature termination codons and in genotoxic stress
responses to DNA damage. We report here a novel role for hSMG-1 in cytoplasmic stress granule (SG)
formation. Exposure of cells to stress causing agents led to the localization of hSMG-1 to SG, identified by
colocalization with TIA-1, G3BP1, and eIF4G. hSMG-1 small interfering RNA and the PIKK inhibitor
wortmannin prevented formation of a subset of SG, while specific inhibitors of ATM, DNA-PKcs, or mTOR had
no effect. Exposure of cells to H2O2and sodium arsenite induced (S/T)Q phosphorylation of proteins. While
Upf2 and Upf1, an essential substrate for hSMG-1 in NMD, were present in SG, NMD-specific Upf1 phos-
phorylation was not detected in SG, indicating hSMG-1's role in SG is separate from classical NMD. Thus, SG
formation appears more complex than originally envisaged and hSMG-1 plays a central role in this process.
Cells are exposed to a variety of genotoxic stresses that
impact on DNA integrity, gene regulation, subcellular organ-
elles, and metabolic events. The phosphoinositide 3 kinase-like
kinase (PIKK), hSMG-1, is an ?400-kDa protein that plays an
important role in cellular viability which is demonstrated by the
embryonic lethality observed in hSMG-1-deficient mice (39).
In addition, hSMG-1 plays a central role in maintaining
mRNA quality through the process of nonsense-mediated
mRNA decay (NMD), where it has been demonstrated to be
crucial for initiating the signaling cascade through phosphor-
ylation of Upf1 at S1078 and S1096, resulting in degradation of
mRNA containing premature termination codons (PTC) (4, 7,
24, 42, 59). PTC-containing mRNAs can be produced through
genomic mutations, alternative splicing, or RNA damage, and
NMD is responsible for the elimination of aberrant PTC-con-
taining mRNAs which could encode nonfunctional truncated
proteins that could interfere with their endogenous counter-
parts (39, 59). NMD is elicited by recognition of the SURF
complex (hSMG-1, Upf1, eRF1, and eRF3) when the termi-
nation codon is situated within ?50bp of the last exon junction
complex (EJC) (7, 23, 24). This results in SMG-1 phosphory-
lating Upf1, leading to NMD-mediated mRNA degradation
(24, 42, 58). Recent work has also implicated two cofactors in
hSMG-1 regulation: SMG-8 and -9 (58). These proteins form
a trimeric complex with hSMG-1 and are required for NMD to
occur. SMG-8 acts to inhibit hSMG-1 kinase activity prior to
interaction with the EJC. In addition to NMD, mRNAs can be
regulated through storage in cytoplasmic stress granules (SG)
or by degradation in the related and often associated struc-
tures, processing bodies (P bodies) (3, 12, 13, 31). SG are
formed in response to cellular stress such as heat shock and
oxidative stress that results in the phosphorylation of eukary-
otic translation initiation factor 2? (eIF2?) (32). SG are com-
posed of accumulated mRNA and their associated proteins,
such as TIA-1, eIF4G, and G3BP1 (32, 33). That SG are only
transiently formed suggests that they are active sites where
individual mRNAs are processed for storage, translation dur-
ing stress and recovery, or shuttled to the associated structures,
PB, for degradation (3, 28, 49).
Brumbaugh et al. (7) and Gewandter et al. (19) demon-
strated that hSMG-1 is a genotoxic stress-activated protein
kinase that displays some functional overlap with the related
kinase, ATM. Expression of hSMG-1 was required for optimal
activation of p53 in response to ionizing radiation (IR) and
small interfering RNA (siRNA) depletion of hSMG-1 caused
constitutive activation of p53 and Chk2, leading to an in-
creased sensitivity to IR (7). As in the case of NMD, Upf1 was
shown to be a substrate for hSMG-1 in response to radiation
damage. hSMG-1 has also been shown to regulate the G1/S
checkpoint in response to prolonged oxidative stress by p53
activation and p53-independent proteolysis of p21 (18).
hSMG-1 also plays a role in telomere stability. Telomeric re-
peats are transcribed into noncoding RNA known as TERRA.
hSMG-1 negatively regulates TERRA association with telo-
meres, and hSMG-1 depletion increased the number of
TERRA-positive chromosomes and resulted in telomere desta-
bilization (6, 9). In addition, depletion of hSMG-1 in tumor
cells markedly increased the extent and accelerated the rate of
apoptosis induced by tumor necrosis factor alpha (TNF-?)
(46). Furthermore, hSMG-1 was demonstrated to be required
for granzyme B-mediated apoptosis in a primary tumor cell
* Corresponding author. Mailing address: Queensland Institute of
Medical Research, 300 Herston Road, Herston, Queensland 4006,
Australia. Phone: 617 3362 0341. Fax: 617 3362 0106. E-mail: martin
† J.A.L.B. and T.L.R. contributed equally to this study.
?Published ahead of print on 12 September 2011.
Page 2
line (41). Inactivation of smg-1 has also been shown to increase
the life span of Caenorhabditis elegans, which appears to be
related to resistance to oxidative stress (38). hSMG-1 has also
been shown to negatively regulate hypoxia-inducible factor 1?
(HIF-1?) in part by blocking mitogen-activated protein (MAP)
kinase activation (10). Thus, it is evident that hSMG-1 is a key
player not only in NMD but also functions in the DNA damage
response, oxidative stress response, hypoxia, and apoptosis.
We describe here a new role for hSMG-1 in the formation of
SG. This role appears to be separate from its role in active
NMD, since although we demonstrated that Upf1 localized to
SG, Upf1 was not phosphorylated on residues known to play a
key role in NMD. hSMG-1 colocalized with a number of SG-
specific markers, and knockdown by siRNA prevented SG for-
mation. Inhibition of PIKK activity by wortmannin treatment
reduced SG formation similar to hSMG-1 knockdown, but
overexpression of kinase dead hSMG-1 did not prevent SG
formation. Our data point to a novel and complex role for
hSMG-1 in SG formation as part of the stress response.
Cell culture conditions. Cells were cultured in RPMI 1640 (lymphoblastoid
cell lines [LCLs]) or Dulbecco modified Eagle medium supplemented with 10%
fetal calf serum (Invitrogen), 100 U of penicillin (Invitrogen)/ml, and 100 U of
streptomycin (Invitrogen)/ml. Primary normal foreskin fibroblasts (NFF) were
established from primary tissue (Glen Boyle, Queensland Institute of Medical
Research). Approximately 11 different NFF strains, obtained from separate
donors, were used during this study. Primary keratinocytes were supplied by N.
Saunders (University of Queensland), and human kidney proximal tubular cells
were supplied by C. Percy (University of Queensland). Primary melanocytes were
also supplied by G. Boyle. The cell lines used were HeLa, U2OS, and A549 and
LCLs. All cells were maintained at 37°C in a humidified atmosphere of 5% CO2
and 95% air.
IR treatment of cells. Irradiation of cells was performed at room temperature
using a137Cs source delivering gamma rays at a dose rate of 1.096 Gy/min (MDS
Gammacell irradiator; Nordion, Ottawa, Ontario, Canada).
Hydrogen peroxide treatment of cells. Adherent cell lines were washed twice
in serum-free medium. The cells were resuspended in 1? original culture volume
of serum-free medium containing H2O2(at the indicated concentrations) and
returned to the incubator.
Heat shock treatment of cells. Adherent cells were placed in a 45°C water bath
and allowed to equilibrate for 5 min. Cells were held at this temperature for 1 h
prior to fixation.
Kinase inhibitor treatment of cells. Wortmannin, AMA-37, and Ku55933
(Calbiochem, La Jolla, CA) were dissolved in dimethyl sulfoxide (DMSO). Ad-
herent cells were grown to the desired density (?70% confluent), and inhibitors
were added directly into the cell culture medium at final concentrations of 10 ?M
for wortmannin and Ku55933 and 30 ?M for AMA-37. Rapamycin (a gift from
Dianne Watters, Griffith University) was used at 20 ?M. Cells were returned to
the incubator for 2 h. Inhibitor treatment was continued in conjunction with any
second agent.
NaAs treatment of cells. Sodium arsenite (NaAs) was dissolved in water to
create a 1 M stock solution. NaAs was added directly into the cell culture
medium at a final concentration of 1 mM for up to 1 h.
Antibodies. Constructs encoding fragments of hSMG-1 fused to glutathione
S-transferase (GST; antibody 1 [Ab1] against amino acids [aa] 24 to 101, Ab2
against aa 155 to 222, and Ab3 against aa 2458 to 2851) were generated in
pGEX5. Recombinant protein production and immunization of sheep was per-
formed as described previously (15). Antibodies were purified from sheep sera by
affinity chromatography. Sera were diluted 1/10 in phosphate-buffered saline
(PBS) and passed over a GST column to deplete the anti-GST antibodies. Eluted
sera were then passed over GST–hSMG-1 columns to isolate anti-hSMG-1 an-
tibodies (the columns were produced using the same protein fragments used for
immunization or a fragment including the N-terminal region detected by Ab1
and Ab2, aa 24 to 222). hSMG-1-specific antibodies were eluted, buffer ex-
changed, and concentrated into a final buffer of PBS, 50% glycerol, and 0.1%
sodium azide. hSMG-1 antibodies were used for immunofluorescence at a 1/100
to 1/600 dilution. hSMG-1 Ab3 was used for Western blotting at 1/1,000. Upf1
was detected using anti-Upf1 antibody (rabbit serum) and Upf2 detected with
anti-Upf2 (affinity-purified rabbit antibody [a gift from L. E. Maquat, University
of Rochester, Rochester, NY]). The phospho-UPF1 antibody that recognizes
pS1078 and pS1096 was supplied by S. Ohno and has been described previously
(59). The phosphorylated (S/T)Q motif antibody (rabbit serum) at a dilution of
1/100 to 1/400 was generated in Robert. T. Abraham's Laboratory at the Burn-
ham Institute for Medical Research (La Jolla, CA). Two batches of this antibody
were used; although the staining intensity differed between the batches of anti-
body, the overall pattern of staining was consistent. The commercial antibodies
and the respective dilutions used for immunofluorescence were as follows: goat
polyclonal TIA-1 antiserum (C20, Sc-1751; Santa Cruz Biotechnology), 1/100;
G3BP1 antibody clone 23 (catalog no. 611126; BD Biosciences), 1/100; rabbit
anti-eIF4G antibody (Santa Cruz catalog no. SC11373, H300), 1/500; and mouse
anti-?H2AX (Upstate Biotechnology). HA was detected by using mouse anti-
hemagglutinin (anti-HA; Covance) at 1/2,000. The antibodies used for Western
blotting were mouse anti-ATM (Genetex) at 1/2,000, rabbit anti-phospho-1981
ATM (R&D Systems) at 1/2,000, mouse anti-DNA-PKcs(Oncogene/Merck) at
1/1,000, and rabbit anti-phospho-2056 DNA-PKcs(Abcam) at 1/2,000.
Conditions for immunofluorescent detection of hSMG-1. Cells were fixed in
1% paraformaldehyde–PBS, followed by permeabilization in 0.1% Triton X-100
and PBS. Nonspecific sites were blocked using 10% fetal calf serum in PBS. All
primary antibodies diluted in 1% newborn calf serum in 0.1% Triton X-100 and
PBS. Alexa Fluor 488- or Alexa Fluor 594-conjugated (Molecular Probes) sec-
ondary antibodies were used at 1/200 to 1/1,000. DNA was visualized using
Hoechst 3339 or DAPI (4?,6?-diamidino-2-phenylindole). Images were captured
using a digital camera (Zeiss AxioCam MRm) attached to a fluorescence micro-
scope (Zeiss Axiovert 2 Mot Plus) with a ?63/1.4 Zeiss Plan-Apochromat oil lens
or using a DeltaVision fluorescence microscopy system (Applied Precision) with
?60/1.4 Olympus Plan-Apochromat oil lens, and images were analyzed with
Softworx imaging software. Deconvolution was performed for five cycles, using a
conservative setting with high noise filtering (Applied Precision). All images
were acquired at room temperature. Colocalization analysis was performed using
Softworx imaging software. Line profiles were determined by drawing a line
through the cytoplasm of SG-positive fibroblasts. Pearson correlation coefficients
(PCCs) were determined for a defined region of the cytoplasm of SG-positive
cells. Between five and ten measurements were performed for each of these
analyses, and representative data are shown.
Mammalian expression vectors. Portions (5 ?g) of plasmid DNA (pSR-HA-
hSMG-1, pSR-HA-hSMG-1-DA, or pSR-HA-Upf1-4SA) (24, 59) were trans-
fected into the indicated cells in a six-well plate using Lipofectamine 2000
(Invitrogen) according to the manufacturer's instructions. The following day,
transfected cells were replated onto coverslips in a 24-well plate and allowed to
adhere for 6 to 8 h prior to treatment with 6 mM sodium butyrate for ca. 16 h
prior to stress treatment. At approximately 48 h posttransfection, the cells were
either left untreated or subjected to various treatments and then rinsed once with
PBS before being fixed in 4% (wt/vol) paraformaldehyde for 10 min. The cells
were then permeabilized in 0.5% (vol/vol) Triton X-100 in PHEM buffer (60 mM
PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2[pH 6.9]) for 10 min. After
a brief rinse in PBS, the cells were blocked in 5% (vol/vol) normal horse serum
plus PBS or fetal calf serum plus PBS for 1 h. The remainder of the staining
protocol was performed as described above.
siRNA knockdown. hSMG-1 and Upf1 stealth siRNA duplex oligoribonucle-
otides were obtained from Invitrogen (hSMG-1-HSS118096-8; Upf1 HSS109171-
3). The duplex oligoribonucleotides were resuspended to a concentration of 20
?M. NFF were grown to ca. 70% confluence prior to use. The volumes of
reagents listed are per well for a six-well plate. siRNA (120 pmol) was added to
final volume of 400 ?l of Opti-MEM (Invitrogen) at 37°C, followed by mixing.
Concurrently, 20 ?l of Lipofectamine 2000 (Invitrogen) was added to 400 ?l of
Opti-MEM, followed by mixing. Each mixture was incubated at room tempera-
ture for 5 min, after which the two mixtures were combined and incubated at
room temperature for a further 20 min. While the mixture was incubating, each
well was washed twice with serum-free medium. After the final wash, 400 ?l of
Opti-MEM and 400 ?l of transfection mix were added to the well. The plate was
returned to the incubator for 6 h, followed by the addition of 2 ml of medium. At
approximately 48 h (hSMG-1) or 64 h (Upf1) after the treatment had com-
menced, the cells were used for assays.
Statistical analysis. Statistical analysis was performed using GraphPad Prism
5 software. The samples were analyzed using a paired two-tailed t test. Statisti-
cally significant differences are marked with asterisks in the figures (*, P ? 0.05;
Sample preparation for and analysis by MS. Samples were processed as
described previously (35). Briefly, samples were separated by SDS-PAGE, incu-
bated in fixing solution (40% ethanol, 10% acetic acid, 50% H2O), and re-
Page 3
buffered in sensitizing solution (30% ethanol, 6.8% [wt/vol] sodium acetate, 0.5%
[wt/vol] sodium thiosulfate), followed by washing. The gel was soaked in silver
solution (0.25% [wt/vol] silver nitrate, 0.015% formaldehyde) and briefly washed
with developing solution (2.5% [wt/vol] sodium carbonate, 0.0074% formalde-
hyde). The reaction was terminated by the addition of stop solution (1.46%
[wt/vol] EDTA). The bands were excised, and the silver stain was removed prior
to tryptic digestion using an adapted form of the method of Gharahdaghi et al.
(20); the method for tryptic digestion was modified from that of Shevchenko et
al. (50). The pellet returned from tryptic digest was resuspended in 5 ?l of 50%
acetonitrile and 0.1% trifluoroacetic acid. This was then used for mass spectro-
photometric (MS) analysis.
For protein identification, peptides were analyzed using a Microflex MALDI-
TOF-PSD (Bruker Daltonics, Bremen, Germany) operated in positive-ion re-
flectron mode. MS data were acquired using 350 shots of a nitrogen laser at 355
nm with a 20-Hz repetition rate and various intensities. The MS data were
calibrated via close external calibration using peptide standards (New England
Biolabs) containing angiotensin I (MH?1,296.69), neurotensin (MH?1,672.92),
ACTH (1 to 17 clip, MH?2,093.09), ACTH (18 to 39 clip, MH?2,465.20), and
ACTH (7 to 38 clip, MH?3,657.93). The Mascot search engine and the Homo
sapiens taxonomic subset of the NCBI nonredundant database were used to
identify the MS data. Mass tolerance was set at 150 ppm. Searches took into
account carbamidomethylated cysteine and oxidized methionine. For the pur-
poses of protein identification, no other posttranslational modifications were
Cell cycle analysis. After indicated treatments, the cells were incubated with
propidium iodide (50 ?g/ml; Sigma) and RNase A (100 ?g/ml; Roche) for 30 min
before cell cycle profiles were quantified using a FACScan (BD Biosciences).
Analysis was performed using ModFit LT (BectonDickinson).
Western blotting. Protein samples analyzed on either hand-poured 4.2% poly-
acrylamide gels or on 4 to 12% Bis/Tris gradient gels (Invitrogen). The proteins
were then transferred onto nitrocellulose membrane, and Western blotting was
performed as described previously (47).
hSMG-1 characterization. We developed three different an-
tibodies that recognize hSMG-1 with a view to investigating
hSMG-1 function in the stress response. The regions of
hSMG-1 recognized by the three antibodies are shown in Fig.
1A. Immunoblot analysis revealed that the antibodies recog-
nized a protein of the expected size for hSMG-1 in extracts
from NFF and LCLs. For all three antibodies, a single high-
molecular-weight band was observed (Fig. 1B), though follow-
ing further separation multiple isoforms of hSMG-1 could be
detected (see Fig. 5A; also data not shown). The bands were
confirmed to be hSMG-1 using immunoprecipitation, followed
by MS; of the 35 peptides identified from tryptic digests, 25
matched hSMG-1, resulting in 7% protein coverage (data not
shown). The specificity of the antibodies was further confirmed
by using siRNA knockdown of hSMG-1 that resulted in a loss
of signal for both immunoblotting and immunostaining (see
Fig. 5A and B). Cellular fractionation followed by immuno-
blotting confirmed that hSMG-1 is present in both the cyto-
plasm and the nucleus (Fig. 1C), as demonstrated previously
(7, 24). Detection of ATM only in the nuclear fraction dem-
onstrated that nuclei were intact. Immunostaining of primary
cells also showed the presence of hSMG-1 in both subcellular
compartments (Fig. 1D, untreated). In response to genotoxic
stress, other members of the PIKK family, including ATM and
ATR (ATM and Rad3 related), localize to nuclear foci at sites
of DNA damage (1, 17). Exposure of primary fibroblasts to IR
gave rise to ?H2AX foci at sites of DNA damage, but there was
no evidence that hSMG-1 localized to these sites under these
conditions (Fig. 1D). However, in a small percentage of cells
(up to 10%) hSMG-1 localized to cytoplasmic granules in re-
sponse to treatment with the DNA-damaging agent hydrogen
peroxide (H2O2) (Fig. 1E). These granules were detected by all
three anti-hSMG-1 antibodies after H2O2treatment (Fig. 1F).
hSMG-1 localization to SG. In fibroblasts, three major types
of cytoplasmic granules have been described: vaults, processing
bodies (P bodies) and SG. These structures are involved in
regulation of translation, mRNA degradation and in stabiliza-
tion and intracellular transport of mRNA (31–33, 49). Of these
structures, only SG are significantly induced by stress-inducing
agents (31). SG form in response to protein translation inhi-
bition and contain the 48S complex and mRNA bound to
T-cell internal antigen 1 (TIA-1) and many other proteins (19,
32). These granules contain proteins that either promote
mRNA stability or lead to destabilization and degradation of
specific mRNA (2). hSMG-1-positive granules, induced by
treatment with H2O2, showed colocalization with TIA-1, indi-
cating that these were indeed SG (Fig. 2A). SG were not
detected with preimmune serum, copurified anti-GST antibody
or in the absence of primary antibody (data not shown). A
commonly used agent to induce SG is sodium arsenite (NaAs)
(30). NaAs causes oxidative stress in cells, increases eIF2?
phosphorylation, and causes translational arrest (2, 37). Expo-
sure of cells to NaAs gave rise to SG, as determined by eIF4G
and hSMG-1 staining (Fig. 2B). Heat was also investigated as
an SG-inducing agent since it induces SG containing unique
SG components (14, 29). Heat treatment induced hSMG-1-
positive SG (Fig. 2B). SG induction in response to NaAs and
heat occurred in at least 70% of cells (see Fig. 5C). Further
confirmation of granule induction was carried out with over-
expressed HA-tagged hSMG-1. Due to the difficulty in trans-
fecting primary cell lines, transient transfection of HeLa cells
with HA-hSMG-1 was performed. SG detected by anti-eIF4G
antibody revealed colocalization with HA-hSMG-1 in SG after
heat shock and H2O2treatment (Fig. 2C). To confirm that the
hSMG-1-positive structures were SG, we used another SG
marker, G3BP1 (52). G3BP1 and hSMG-1 colocalized after
treatment with heat (Fig. 2D). The number of SG per cell and
the number of SG-positive cells varied (particularly with
H2O2), and these granules showed a great deal of heterogene-
ity in size (data not shown), findings consistent with previous
observations (27, 31).
The universality of SG induction in other primary cells was
also investigated. Figure 3A shows merged images of hSMG-1
(red) and TIA-1 (green) staining for SG induced in kidney
proximal tubular cells, melanocytes, and human undifferenti-
ated keratinocytes. With both H2O2and heat treatment,
hSMG-1 and TIA-1 colocalized for all of the primary cells. In
contrast, the carcinoma cell lines U2OS and A549 were unable
to form hSMG-1 staining granules but formed TIA-1-positive
granules after treatment with NaAs (Fig. 3B).
Since hSMG-1 has an established role in NMD, we investi-
gated the possibility that its localization to SG was connected
with NMD and/or mRNA decay. Recruitment of hSMG-1 in
this process is generally mediated by Upf2 and Upf3b stably
associated with the EJC (24). Therefore, we determined
whether Upf2 was localized to SG. Upf2 localized with TIA-1
to SG in response to heat (Fig. 4A). Another key member of
this mRNA surveillance complex, Upf1, was also present in SG
induced by H2O2, heat, and NaAs, as shown by colocalization
with eIF4G or hSMG-1 (Fig. 4B and C). This colocalization
was quantified, yielding a PCC of 0.7832, where a PCC value
VOL. 31, 2011 ROLE FOR hSMG-1 IN STRESS GRANULE FORMATION4419
Page 4
between 0.5 and 1 is considered a positive association. A line
profile of fluorescence in the red and green channels also
confirmed the overlapping signals (Fig. 4D). A key step in the
targeting of NMD is phosphorylation of Upf1 at four specific
sites by hSMG-1 (S1073, S1078, S1096, and S1116), which
appears to be critical for recognition of the PTC (24). A Upf1
phospho-specific antibody recognizing pS1078 and p1096
failed to detect phosphorylation in SG, suggesting that Upf1
phosphorylation is not required for SG formation or that it
occurs at different sites (Fig. 4E). The localization of a form of
Upf1 (HA4SAUpf1), nonphosphorylatable at all four sites,
confirms the findings obtained with the P-Upf1 antibody (Fig.
4F). These observations are in keeping with a report by Gard-
ner (16), which showed that, in response to hypoxia and arse-
nic, Upf1 localized to SG and NMD was inhibited (16). How-
ever, hSMG-1 mediated NMD-dependent phosphorylation at
the remaining 23 predicted PIKK phosphorylation sites
not be ruled out. To determine whether hSMG-1 could be
regulating another form of mRNA decay, we determined
FIG. 1. hSMG-1 is cytoplasmic and nuclear and localizes to cytoplasmic granules in response to stress. (A) Schematic of the regions recognized
by of hSMG-1 antibodies. GST fusion proteins were prepared using the regions indicated (region 1, Ab1; region 2, Ab2; region 3, Ab3) and used
to generate polyclonal antibodies in sheep. (B) Specificity of detection of hSMG-1 by immunoblotting. Extracts were prepared from LCLs and
NFF, and proteins were separated by SDS-PAGE prior to immunoblotting with the three different hSMG-1 antibodies. (C) hSMG-1 is present both
in the cytoplasm and in the nucleus. Immunoblot analysis was performed for hSMG-1 in nuclear and cytoplasmic extracts from primary normal
foreskin fibroblasts (NFF). ATM was used as a control to demonstrate fractionation. (D) hSMG-1 does not localize to sites of DNA damage. DNA
damage induced nuclear foci in response to 10 Gy of IR. NFF were fixed and stained with anti-?H2AX (red) and anti-hSMG-1 (Ab2, green)
antibodies. Nuclei were detected with DAPI. (E) Detection of cytoplasmic granules in NFF in response to H2O2. The cells were incubated for 1 h
after exposure, fixed, and stained with hSMG-1 (Ab2) antibody. (F) Immunofluorescent detection of hSMG-1 with three different antibodies in
NFF. hSMG-1 antibodies (red) directed to different regions of hSMG-1 show cytoplasmic granule formation in response to heat.
Page 5
whether hSMG-1 localized to P bodies. We used a construct
encoding the P-body-specific marker decapping protein 1
(DCP1), which is required for mRNA decapping and degra-
dation (49). No significant colocalization between hSMG-1-
and DCP-1-positive granules was observed after treatment of
fibroblasts with H2O2, providing further confirmatory evidence
that hSMG-1 was localized specifically to SG under these con-
ditions (Fig. 4G).
hSMG-1 is required for NaAs-induced SG formation or
maintenance. To further establish the importance of hSMG-1
in SG formation, three different siRNA duplexes were used to
reduce cellular levels of hSMG-1. In these experiments we
focused on heat and NaAs as inducing agents because they
reproducibly induced SG in a high percentage of cells. Figure
5A shows that all siRNA sequences significantly reduced but
did not ablate hSMG-1 protein expression. After siRNA treat-
ment of fibroblasts, SG formation was decreased after NaAs
treatment but not after exposure to heat (Fig. 5B). The per-
centage of cells containing eIF4G-positive SG was the same
after heat treatment but was significantly reduced in NaAs-
treated cells (Fig. 5C). siRNA knockdown of hSMG-1 also
appeared to decrease SG formation in response to H2O2(data
not shown), although the very low and variable percentage of
cells forming SG in response to H2O2meant these data were
not statistically significant. These data point to an important
role for hSMG-1 in the formation or stability of a subgroup of
SG, although more complete inhibition of SG formation may
occur if total hSMG-1 knockdown was achieved. This experi-
ment also demonstrates that heat-induced SG formation is
hSMG-1 independent. To further examine the role NMD
might play in SG regulation, we knocked down Upf1 expres-
sion with siRNA (Fig. 5D). As determined by Western blot-
ting, the protein level of Upf1 was decreased by two indepen-
dent siRNA sequences. The Upf1 siRNA treatment did not
affect the protein levels of either hSMG-1 or TIA-1 (Fig. 5D).
siRNA treatment in this experiment did not completely knock
down Upf1 expression; however, if the amount of siRNA used
or the length of incubation was increased, fibroblast cell via-
bility was severely compromised (data not shown). Therefore,
we used these conditions to examine the role of Upf1 in SG
formation. After siRNA knockdown of Upf1, the fibroblasts
were treated with either NaAs or heat for 1 h to induce SG
formation. The cells were then immunostained for Upf1 and
TIA-1, and SG formation was quantified. Knockdown of Upf1
showed a small decrease in SG formation in response to both
NaAs and heat, although this was not statistically significant
(data not shown). However, in many cells treated with anti-
Upf1 siRNA, sufficient Upf1 protein was present to see it
recruited to SG (Fig. 5E). Decreased SG formation after Upf1
knockdown was not due to inhibition of hSMG-1 recruitment
to SG since hSMG-1 clearly localized in SG even after Upf1
depletion (Fig. 5F). Therefore, whether Upf1 plays a direct
role in SG formation or whether depletion of Upf1 generally
suppresses cellular responses is currently unclear.
FIG. 2. hSMG-1 localizes to stress granules. (A) hSMG-1 (detected by Ab1, red) colocalizes with the SG marker TIA-1 (green) in response to
H2O2. NFF were stained after H2O2treatment (1.5 mM) for 1 h. (B) Colocalization of hSMG-1 (Ab2, red) with a second SG marker eIF4G (green)
after treatment of NFF with heat (45°C) or 1 mM NaAs for 1 h. DAPI (blue) was used to stain nuclei. Colocalization (yellow) is indicated in a
merged panel on the right. (C) An HA-tagged form of hSMG-1 localizes to SG. HeLa cells were transiently transfected with HA-hSMG-1 and
incubated for 48 h in fresh medium prior to exposure to heat or H2O2, followed by staining with anti-HA or anti-eIF4G antibodies. (D) hSMG-1
(Ab1) (red) colocalizes with G3BP1 (green) in SG formed in NFF in response to heat. Hoechst was used to stain the nuclei.
VOL. 31, 2011 ROLE FOR hSMG-1 IN STRESS GRANULE FORMATION4421
Page 6
Role of hSMG-1 kinase activity in SG formation. hSMG-1
may be required for SG formation for either of two reasons: (i)
hSMG-1 kinase activity may be required for formation or sta-
bility of SG or, (ii) alternatively, the hSMG-1 protein may
physically be required. In common with other members of the
PIKK family, hSMG-1 phosphorylates substrates at (S/T)Q
motifs in response to DNA damage (7, 59). Since there is no
specific hSMG-1 inhibitor, we investigated the role of hSMG-1
kinase activity in SG formation using the PIKK inhibitor wort-
mannin (48). Wortmannin exposure for 2 h prior to stress
treatment blocked SG formation after treatment with NaAs
but did not inhibit SG formation induced by heat treatment
(Fig. 6A). This further supported the hSMG-1-independent
formation of heat-induced SG previously observed (Fig. 5B
and C). Wortmannin appeared to also inhibit SG formation in
the small number of cells that responded to H2O2(data not
shown). To investigate whether inhibition of other PIKK fam-
ily members may have resulted in the observed decrease in SG
formation, specific inhibitors of the related PIKK family mem-
bers were used. ATM (Ku55933) and DNA-PK (AMA-37)
FIG. 3. hSMG-1 plays a central role in SG formation in primary cells but does not localize to SG in tumor cells. (A) Appearance of SG in
primary cells in response to H2O2or heat treatment. The images represent merged images between hSMG-1 (Ab1) (red) and TIA-1 (green). Nuclei
were visualized with Hoechst stain. Abbreviations: PTC, human proximal tubular cells; MEL, primary melanocytes; HEK, undifferentiated
keratinocytes. (B) A549 and U2OS cells were treated with NaAs for 1 h and stained for hSMG-1 (Ab1) (red) and TIA-1 (green). TIA-1-positive
but hSMG-1-negative SG were formed in response to NaAs.
Page 7
FIG. 4. Localization of NMD proteins to SG but lack of evidence for a role for hSMG-1 in mRNA decay. (A) Upf2 colocalizes with TIA-1 in
SG. NFF were fixed with or without heat treatment and stained for TIA-1 (green) and Upf2 (red). Colocalization (yellow) following heat treatment
is shown in the merged panel on the right. (B) Upf1 also localizes to SG. HeLa cells were transiently transfected with HA-Upf1. After 48 h, the
cells were treated with H2O2and NaAs and then stained with anti-HA (green) and anti-eIF4G (red) antibodies. (C) Endogenous Upf1 localizes
to hSMG-1. NFF were treated with NaAs or heat and after 1 h stained for Upf1 (green) and hSMG-1 (Ab2, red). (D) Upf1 colocalizes with
hSMG-1. The line profile shows overlapping fluorescence signals for Upf1 (green) and hSMG-1 (red). Image analysis was performed using the
Softworx computer program. Line profiles were determined by drawing a line through the cytoplasm of SG positive fibroblasts. (E) Upf1 is not
phosphorylated in heat- and NaAs-induced SG. NFF were treated with NaAs or heat and, after 1 h, stained with phospho-specific antibodies
recognizing sites phosphorylated on Upf1 during NMD. P-Upf1 (green) was not detected in hSMG-1 (Ab2, red) positive SG but could be clearly
seen in the nucleus. (F) Localization of a nonphosphorylatable form of Upf1, HA4SAUpf1, to SG. NFF were transiently transfected with
HA4SAUpf1 and incubated for 48 h in fresh medium prior to exposure to H2O2and heat, followed by staining with anti-HA and anti-Upf1
antibodies. (G) hSMG-1 (Ab1) does not localize to P bodies. NFF were transiently transfected with the P-body-specific marker DCP1-YFP and
incubated for 48 h prior to treatment with H2O2for 1 h and followed by the detection of YFP and staining with antibodies against hSMG-1.
VOL. 31, 2011 ROLE FOR hSMG-1 IN STRESS GRANULE FORMATION4423
Page 8
inhibitors did not significantly inhibit SG formation in response
to either NaAs or heat treatment (Fig. 6A). We also checked
for the involvement of another member of the PIKK family
mammalian target of rapamycin (mTOR) that controls cell
growth and survival (51). Inhibition of mTOR by rapamycin
also failed to interfere with heat- or NaAs-induced SG (Fig.
6A). All inhibitors were shown to be active under these con-
ditions, as evidenced by the inhibition of radiation-induced
FIG. 5. Effect of hSMG-1 or Upf1 disruption on SG formation. (A) Knockdown of hSMG-1 in NFF by three different siRNA oligonucleotides.
hSMG-1 protein was detected by immunoblotting (Ab3), and the loading control was DNA-PKcs. (B) At 48 h after siRNA treatment, the NFF were
treated with NaAs or heat for 1 h and then stained for hSMG-1 (Ab2, red) and eIF4G (green). Colocalization appears as yellow. Nuclei were
detected with DAPI (blue). After siRNA treatment, the numbers of eIF4G SG-positive cells were reduced in NFF that had been treated with
NaAs but not in heat-treated NFF. Control siRNA had no affect on SG formation in response to either agent. (C) Quantification of SG
formation following treatment with two different anti-hSMG-1 or control siRNA. The percentage of NFF with eIF4G-positive granules was
scored. The data represent the averages of three independent experiments, and error bars show the standard errors of the mean. Asterisks
denote statistically significant differences in SG formation (*, P ? 0.05;**, P ? 0.01). (D) Knockdown of Upf1 expression in NFF using two
different siRNA. Western blotting confirmed knockdown of Upf1 protein level but no affect on hSMG-1 or TIA-1 expression levels. GAPDH
was probed for as a loading control. An irrelevant lane has been removed from the image, but all samples were run on the same gel. (E) Upf1
knockdown does not block stress granule formation. Approximately 64 h after anti-Upf1 siRNA treatment SG were induced with either NaAs
or heat for 1 h. Fibroblasts were stained for TIA-1 (green) and Upf1 (red). SG formation could be seen in all treated samples. The data are
representative of three independent experiments. (F) Cells were treated as for panel E but stained for hSMG-1 (green) and Upf1 (red).
Nuclei were visualized with DAPI (blue). hSMG-1 was still recruited to SG following Upf1 knockdown. The data are representative of three
independent experiments.
Page 9
ATM autophosphorylation measured by S1981 phosphoryla-
tion, inhibition of DNA-PKcsautophosphorylation on S2056,
and rapamycin inhibition of mTOR, which blocked the move-
ment of cells from G1into S phase (Fig. 6B to D). Quantifi-
cation of the immunofluorescent images showed far less colo-
calization after wortmannin treatment than after treatment
with any other inhibitor (a PCC of 0.8082 for DMSO versus a
PCC of 0.4798 for wortmannin) (Fig. 6E). These results sug-
gest that PIKK activity is essential for SG formation in re-
sponse to NaAs but not to heat.
We looked for the presence of potential PIKK substrates in
SG by staining with a phospho-specific antibody against p(S/
T)Q sites. In response to NaAs and heat treatment, speckles of
p(S/T)Q staining could be observed in the cytoplasm of cells
(Fig. 7A). These sites were not completely colocalized with
hSMG-1 but were often overlapping or associated with hSMG-
1-positive granules (PCC of 0.4497; see also the left panel of
Fig. 7B). In contrast, SG formed in response to heat were
strongly hSMG-1 positive, but no phosphorylated (S/T)Q sites
were detected (Fig. 7A, lower panel). In response to H2O2, a
much stronger phosphorylated (S/T)Q signal, more completely
colocalizing with hSMG-1, was detected (PCC of 0.6988) (Fig.
7A, right panel of Fig. 7B). These data imply that PIKK activity
may be important in the regulation or stability of NaAs- and
H2O2-induced SG but not heat-induced SG.
To further investigate the specific requirement of hSMG-1
FIG. 6. Role for PIKK kinase activity in SG formation. (A) Wortmannin inhibits SG formation in response to NaAs treatment but not heat
treatment. Cells were NaAs or heat treated after 2 h of incubation with the indicated inhibitor. NFF were then stained with anti-hSMG-1 (Ab2,
red) and anti-eIF4G (green) antibodies, with the colocalization appearing as yellow. Nuclei were detected with DAPI (blue). Inhibition of ATM
(Ku55933), DNA-PKcs(AMA37), or mTOR (rapamycin) did not interfere with formation of SG in response to either treatment. The data are
representative of two to four independent experiments. (B) Effect of ATM-specific inhibitor on ATM autophosphorylation at S1981. NFF were
incubated with Ku55933 for 2 h prior to 10 Gy of IR and then incubated for 30 min or 1 h prior to the preparation of cell extracts. Immunoblotting
was carried out with a phospho-specific antibody for ATM S1981. Nbs1 protein was used as a loading control. (C) Effect of different inhibitors on
DNA-PK autophosphorylation at S2056. NFF were incubated with either Ku55933 (ATM inhibitor), wortmannin (general PIKK inhibitor), or
AMA37 (DNA-PKcsinhibitor) for 2 h prior to exposure to 10 Gy of IR. Extracts were prepared and immunoblotted either with the phospho-
specific antibody (P2056-DNA-PK) or an anti-DNA-PKcsantibody. Total DNA-PKcsdetection was used as a loading control. (D) Rapamycin
prevents passage of NFF from G1to S phase. Cells were exposed to concentrations of rapamycin from 20 to 100 ?M prior to analysis by flow
cytometry using propidium iodide (PI) staining. (E) PCCs for cells treated with PIKK inhibitors prior to SG formation. These coefficients were
determined using Softworx software for a defined region of the cytoplasm of fibroblasts. The data show the averages of at least five measurements
from different cells from at least two independent experiments and the standard deviations of the measurements.
VOL. 31, 2011 ROLE FOR hSMG-1 IN STRESS GRANULE FORMATION4425
Page 10
kinase activity in SG formation in response to NaAs and H2O2,
a kinase-deficient mutant of hSMG-1 (HA-hSMG-1-DA) was
transiently transfected into HeLa cells. Kinase-dead hSMG-1
localized as efficiently as wild-type hSMG-1 to SG induced by
H2O2or NaAs (Fig. 7C). In SG containing wild-type HA-
SMG-1, phosphorylated (S/T)Q signal was observed in re-
sponse to both H2O2and NaAs (Fig. 7C). In SG containing the
kinase-deficient HA-hSMG-1-DA, phosphorylated (S/T)Q
sites were undetectable in response to H2O2treatment (Fig.
7C), suggesting that hSMG-1 is responsible for the observed
phosphorylation. However, phosphorylated (S/T)Q sites were
still present in NaAs-induced SG containing the kinase-dead
hSMG-1, indicating that another member of the PIKK family
is capable of phosphorylation of these sites. Collectively, the
experiments described here show that hSMG-1 is recruited to
SG and its presence, but not kinase activity, is essential for
NaAs- and H2O2-induced, but not heat-induced, SG forma-
hSMG-1 has been shown to play several distinct roles in the
cellular response to stress, including its involvement in NMD
(59), in the protection of cells against TNF-?- or granzyme
B-induced apoptosis (41, 46), and in activating the G1/S check-
point (18). More recently Chen et al. (10) found that hSMG-1
inhibited HIF-1? transactivation activity in part by suppressing
MAP kinase ERK1 in hypoxia. These results suggested that
hypoxic conditions were efficient in activating hSMG-1 to re-
strain hypoxia-induced malignancy. Furthermore, mutations of
the hSMG-1 kinase domain have been observed in breast can-
cer, lung adenocarcinoma, and kidney and stomach cancer (11,
53,57; http://www.sanger.ac.uk/genetics/CGP/cosmic).
short, SMG-1 has known roles in NMD and genome mainte-
nance and is implicated in regulation of oxidative stress, apop-
tosis and hypoxia responses. We have identified here a novel
role for hSMG-1 in the formation of SG.
FIG. 7. Detection of phosphorylated proteins in SG and importance of protein kinase activity of hSMG-1. (A) NaAs and H2O2treatment of
NFF induced the appearance of proteins phosphorylated at (S/T)Q sites, and these sites partially colocalize in SG with hSMG-1. Heat induced SG
formation (hSMG-1 positive) without detectable phosphorylation at (S/T)Q sites. Staining in the upper panel used an older batch of rabbit
anti-P(S/T)Q antibody: P(S/T)Q (green), hSMG-1 (Ab2, red), and DAPI (blue). In the lower panel, a newer batch of rabbit anti-P(S/T)Q antibody
was used: P(S/T)Q (red), hSMG-1 (Ab2, green), and DAPI (blue). (B) P(S/T)Q signals associate with hSMG-1 after SG induction. The left panel
shows a line profile indicating regions of overlapping fluorescence signals for P(S/T)Q (green) and hSMG-1 (red) following NaAs treatment. The
right-hand panel shows greater overlap of the P(S/T)Q (green) signal with hSMG-1(red) after H2O2treatment. Image analysis was performed using
the Softworx computer program. Line profiles were determined by drawing a line through the cytoplasm of SG-positive fibroblasts. (C) Overex-
pression of kinase-deficient hSMG-1 does not inhibit SG formation. HeLa cells were transfected with either HA-hSMG-1 or HA-hSMG-1-DA
(kinase deficient). The cells were treated with 1 mM H2O2or NaAs for 1 h and then stained with anti-HA (green) and anti-P(S/T)Q (red)
antibodies. In cells transfected with HA-hSMG-1-DA, no P(S/T)Q sites were observed in response to H2O2treatment.
Page 11
SG are related to other mRNA-protein complexes, including
P bodies, germ cell granules, and neuronal RNA granules (8),
all of which contain a variety of RNA-binding proteins, trans-
lation factors, and RNA decay machinery that can change
depending on exposure to cellular stress (31). However, P
bodies and SG are the only types present in fibroblasts. Typi-
cally, SG contain the 48S preinitiation complex composed of
the small ribosomal subunit and mRNA, together with a num-
ber of translation initiation factors, poly(A)-binding protein,
proteins that regulate mRNA translation, and proteins in-
volved in cell signaling pathways (2, 44). SG are commonly
described as structures that sequester RNA during times of
cellular stress either to promote degradation, to stabilize
mRNA for rapid translation once the stress has abated, or to
promote translation of specific mRNA during stress (2). Nas-
cent mRNA transcripts can also be exported from the nucleus
and targeted directly to SG (8). This finding shows that while
SG form in response to translational arrest, not all transcripts
in SG are associated with stalled ribosomal complexes. More
recently, alternative functions for SG have been described. SG
have been shown to harbor proteins involved in the regulation
of apoptosis such as TRAF2, RACK, and FAST (5, 34, 36).
These data suggest that SG act to limit apoptosis, while cells
adapt to stress, since inhibition of SG formation during stress
can lead to decreased cell survival (8). Furthermore, SG have
been implicated in cellular responses to viral infection (8),
although the role SG play here is unclear since many viruses
disrupt or induce SG formation to benefit their own life cycle.
Previous work has shown that in mammalian cells NMD fac-
tors, including Upf1 and NMD mRNA targets, can traffic
through P bodies (12) and localize to SG (16) when NMD is
inhibited by specific inhibitors or in response to hypoxia.
We showed here that hSMG-1 is recruited to SG in response
to a range of cellular stresses and that knockdown of hSMG-1
strongly reduced SG formation in response to NaAs but not to
heat. Although use of the PIKK inhibitor wortmannin pre-
vented SG formation in response to NaAs, overexpression of a
kinase-dead version of hSMG-1 did not. These data suggest
that the role of hSMG-1 in stress responses is likely to be
dependent on the type of cellular stress encountered. There
are potentially three facets to the involvement of hSMG-1 in
SG: (i) the mechanism of hSMG-1 recruitment to SG, (ii) the
requirement for hSMG-1 as a protein facilitating SG formation
following certain stresses, and (iii) the role of PIKK, including
hSMG-1, kinase activity in either SG formation, function, or
Mechanism of hSMG-1 recruitment to SG. Stress-induced
signaling leading to phosphorylation of eIF2? and SG forma-
tion can be initiated by at least four different kinases (GCN2,
PERK, HRI, and PKR) (22, 54). These kinases function in
conjunction with other signaling pathways to coordinate the
cellular response to a specific stress. Some of these pathways,
converging on eIF2?, may have additional parallel effects fa-
cilitating recruitment of hSMG-1 to SG. The recruitment of
hSMG-1 to SG in response to all stresses tested may be linked
to inhibition of NMD. During the response to hypoxia Upf1
localized to SG and under the same conditions NMD was
inhibited, although a causative link between these phenomena
was not established (16). Furthermore, a very recent study
showed that a variety of cellular stresses (including the SG-
inducing agent NaAs) resulted in the phosphorylation of eIF2?
and inhibition of NMD (55). Although in neither study was a
causative link between SG formation and NMD inhibition
established. In addition, a hyperphosphorylated form of Upf1
accumulated in P bodies in response to chemical inhibition of
NMD, which blocks NMD at a step following Upf1 phosphor-
ylation (12). In the present study, we observed the recruitment
of hSMG-1, Upf1, and Upf2 to SG, but Upf1 was not phos-
phorylated at known NMD sites under these conditions, indi-
cating that active NMD was not occurring within SG. This
result is supported by the recent finding (55) that NMD was
inactivated after treatment with the SG-inducing agent NaAs
used here. Since Upf1 detected in P bodies after NMD inhi-
bition was hyperphosphorylated, the unphosphorylated form
we detected in SG may have an NMD independent role, or it
may represent a different form of NMD inhibition more similar
to that observed during hypoxia (12, 16). It is likely that under
all of the stresses examined here, NMD is inhibited (55).
Therefore, the recruitment of hSMG-1 and Upf1 to SG, with-
out being essential for SG formation, may provide the basis of
a mechanism for NMD inhibition in response to the phosphor-
ylation of eIF2? (55). Alternatively, hSMG-1 may be required
to traffic or process specific transcripts in SG under stress
conditions such as heat shock, where it is not essential for SG
Requirement of hSMG-1 as a protein facilitating SG forma-
tion following certain stresses. hSMG-1 clearly plays a role in
the formation or stability of SG after treatment with NaAs or
H2O2but not heat treatment. Knockdown of hSMG-1 with
siRNA reduced the formation of SG in response to these
agents, but a kinase-dead version of hSMG-1 also strongly
localized to SG. This construct has previously been shown to
act in a dominant-negative manner (24). Therefore, the data
suggest that the presence of the hSMG-1 protein, but not its
kinase activity, is required for SG formation in response to
some stresses. This role for hSMG-1 has not been described
before. Using an RNA-mediated interference-based screen,
Ohn et al. (45) identified 100 human genes required for SG
assembly. hSMG-1 was not identified in that screen, which may
be due to differences in the cell lines used or that knockdown
of hSMG-1 induces apoptosis (7), removing these cells from
further analysis. To determine whether the requirement for
hSMG-1 in SG formation was related to NMD, we attempted
to define the role of the hSMG-1 substrate Upf1 in response to
NaAs using siRNA knockdown. However, substantial Upf1
depletion decreased cell viability. This is not surprising since
Upf1 has previously been shown to be required for embryonic
development (40) and another NMD component Upf2 was
shown to be essential for the viability of hematopoietic stem
cells (56). Consequently, the decrease in SG formation ob-
served with Upf1 knockdown can be interpreted in two ways:
(i) Upf1 is required for SG formation in response to both
NaAs and heat, but sufficient Upf1 depletion could not be
achieved in order to see a dramatic effect, or (ii) Upf1 is not
required for SG formation, and the small decrease observed
here may be due to decreased viability or functionality of the
cells. At this stage, it is still unclear what role Upf1 plays in SG
formation. Combined, these data suggest that hSMG-1 is re-
quired for SG formation in response to NaAs in a manner that
may be related to its role in NMD, but its retention in SG is
VOL. 31, 2011ROLE FOR hSMG-1 IN STRESS GRANULE FORMATION 4427
Page 12
independent of its kinase activity. Known roles for hSMG-1 are
associated with its kinase activity. An understanding of this
novel role for hSMG-1 will require further biochemical anal-
ysis of the protein and its stress induced interaction partners
and/or isolation of different classes of SG to see what is unique
about SG where hSMG-1 is required for their formation. Cur-
rently, isolation of SG in this manner is not possible. The
differential requirement of hSMG-1 for SG formation may
reflect the ability of cells to survive after insult by different
stresses or reflect different mechanisms of translation or NMD
inhibition induced (8).
Role of PIKK activity in SG formation, function, or disas-
sembly. The final aspect of hSMG-1 function in SG is the
potential role of PIKK activity in the cellular response to
stress. Phosphorylation of PIKK target (S/T)Q sites was de-
tected in or associated with SG in response to NaAs and H2O2
treatment. Wortmannin inhibition blocked formation of SG in
response to these stresses, indicating that PIKK activity is
essential for SG formation under these conditions. Interest-
ingly, overexpression of a kinase-dead version of hSMG-1 in
HeLa cells did not prevent SG formation and, in fact, the
kinase-dead form localized to SG with an efficiency similar to
that of wild-type hSMG-1 in response to both stimuli, although
this may be due to the ability of hSMG-1 to dimerize (42). To
further complicate the picture, H2O2-induced phosphorylation
of (S/T)Q sites was inhibited by kinase-dead hSMG-1, but
NaAs-induced phosphorylation was not. These data suggest
either that hSMG-1 kinase activity is not involved in SG for-
mation or that hSMG-1 kinase activity can be compensated for
by another PIKK family member in this situation. However,
hSMG-1 kinase activity may be involved in SG regulation in
response to H2O2since hSMG-1-dependent phosphorylation
sites were observed in these SG. Other PIKK family members
have been implicated in SG formation. In the RNAi screen
discussed above, the PIKK family member, DNA-PKcswas
detected, indicating that its kinase activity may also be impor-
tant (45). However, in the presence of a specific DNA-PKcs
inhibitor we did not observe interference with SG formation in
response to either NaAs or heat treatment. We also failed to
prevent SG formation using the ATM-specific inhibitor
KU55933 (21) or rapamycin, an inhibitor of mTOR (51), de-
spite wortmannin strongly inhibiting SG formation. At the
concentration used wortmannin inhibition of activity of pro-
teins other that members of the PIKK family should be mini-
mal. Potentially, multiple PIKK family members may be in-
volved in an overlapping or redundant manner in SG
regulation, as they are in the nuclear DNA damage response.
If so, inhibition of more than one would be required to see a
reduction in SG formation in response to NaAs or H2O2.
Interestingly, PIKK family members have also been impli-
cated in NMD-independent RNA degradation involving Upf1.
Histone mRNA stability may also be controlled by DNA-PK-
mediated phosphorylation of Upf1 (43), and ATR may also
phosphorylate Upf1 during histone mRNA degradation (25,
26). hSMG-1 involvement in this process has not been inves-
tigated. How this process may relate to a role for PIKK in SG
regulation will require further investigation.
Overall, we show that hSMG-1 is recruited to SG in re-
sponse to heat, NaAs, and H2O2treatment. Our data suggest
that the physical presence of the hSMG-1 protein is required
for formation of a subset of SG independently of its protein
kinase activity and that protein phosphorylation by PIKKs,
including hSMG-1, may be involved in the regulation and/or
turnover of SG in response to specific stresses.
We thank Paul Anderson and Nancy Kedersha (Harvard Medical
School) for the DCP1-eYFP construct, L. E. Maquat (University of
Rochester Medical Centre) for the Upf1 and Upf2 antibodies and
constructs, and Dianne Watters (Griffith University) for the rapamy-
cin. We thank Glen Boyle (Queensland Institute of Medical Research)
for the primary fibroblasts, Nicholas Saunders (University of Queens-
land) for the primary keratinocytes, C. Percy (University of Queens-
land) for the human kidney proximal tubular cells, Aine Farrell for her
tissue culture expertise, and the Lavin laboratory for stimulating dis-
cussions. We also thank Tracey Laing for typing the manuscript.
We thank the Australian Research Council for funding and the
National Health and Medical Research Council of Australia for a
Peter Doherty Fellowship to T.L.R.
1. Andegeko, Y., et al. 2001. Nuclear retention of ATM at sites of DNA double-
strand breaks. J. Biol. Chem. 276:38224–38230.
2. Anderson, P., and N. Kedersha. 2006. RNA granules. J. Cell Biol. 172:803–
3. Anderson, P., and N. Kedersha. 2009. Stress granules. Curr. Biol. 19:R397–
4. Arias-Palomo, E., et al. 2011. The nonsense-mediated mRNA decay SMG-1
kinase is regulated by large-scale conformational changes controlled by
SMG-8. Genes Dev. 25:153–164.
5. Arimoto, K., H. Fukuda, S. Imajoh-Ohmi, H. Saito, and M. Takekawa. 2008.
Formation of stress granules inhibits apoptosis by suppressing stress-respon-
sive MAPK pathways. Nat. Cell Biol. 10:1324–1332.
6. Azzalin, C. M., P. Reichenbach, L. Khoriauli, E. Giulotto, and J. Lingner.
2007. Telomeric repeat containing RNA and RNA surveillance factors at
mammalian chromosome ends. Science 318:798–801.
7. Brumbaugh, K. M., et al. 2004. The mRNA surveillance protein hSMG-1
functions in genotoxic stress response pathways in mammalian cells. Mol.
Cell 14:585–598.
8. Buchan, J. R., and R. Parker. 2009. Eukaryotic stress granules: the ins and
outs of translation. Mol. Cell 36:932–941.
9. Chawla, R., and C. M. Azzalin. 2008. The telomeric transcriptome and SMG
proteins at the crossroads. Cytogenet. Genome Res. 122:194–201.
10. Chen, R. Q., et al. 2009. Kinome siRNA screen identifies SMG-1 as a
negative regulator of hypoxia-inducible factor-1? in hypoxia. J. Biol. Chem.
11. Ding, L., et al. 2008. Somatic mutations affect key pathways in lung adeno-
carcinoma. Nature 455:1069–1075.
12. Durand, S., et al. 2007. Inhibition of nonsense-mediated mRNA decay
(NMD) by a new chemical molecule reveals the dynamic of NMD factors in
P-bodies. J. Cell Biol. 178:1145–1160.
13. Eulalio, A., I. Behm-Ansmant, and E. Izaurralde. 2007. P bodies: at the
crossroads of posttranscriptional pathways. Nat. Rev. Mol. Cell. Biol. 8:9–22.
14. Farny, N. G., N. L. Kedersha, and P. A. Silver. 2009. Metazoan stress granule
assembly is mediated by P-eIF2?-dependent and -independent mechanisms.
15. Frangioni, J. V., and B. G. Neel. 1993. Solubilization and purification of
enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal.
Biochem. 210:179–187.
16. Gardner, L. B. 2008. Hypoxic inhibition of nonsense-mediated RNA decay
regulates gene expression and the integrated stress response. Mol. Cell. Biol.
17. Gatei, M., et al. 2001. Ataxia telangiectasia mutated (ATM) kinase and ATM
and Rad3-related kinase mediate phosphorylation of Brca1 at distinct and
overlapping sites: in vivo assessment using phospho-specific antibodies.
J. Biol. Chem. 276:17276–17280.
18. Gehen, S. C., R. J. Staversky, R. A. Bambara, P. C. Keng, and M. A. O'Reilly.
2008. hSMG-1 and ATM sequentially and independently regulate the G1
checkpoint during oxidative stress. Oncogene 27:4065–4074.
19. Gewandter, J. S., R. A. Bambara, and M. A. O'Reilly. 2011. The RNA
surveillance protein SMG1 activates p53 in response to DNA double-strand
breaks but not exogenously oxidized mRNA. Cell Cycle 10:15.
20. Gharahdaghi, F., C. R. Weinberg, D. A. Meagher, B. S. Imai, and S. M.
Mische. 1999. Mass spectrometric identification of proteins from silver-
stained polyacrylamide gel: a method for the removal of silver ions to en-
hance sensitivity. Electrophoresis 20:601–605.
21. Hickson, I., et al. 2004. Identification and characterization of a novel and
Page 13
specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer
Res. 64:9152–9159.
22. Holcik, M., and N. Sonenberg. 2005. Translational control in stress and
apoptosis. Nat. Rev. Mol. Cell. Biol. 6:318–327.
23. Isken, O., and L. E. Maquat. 2008. The multiple lives of NMD factors:
balancing roles in gene and genome regulation. Nat. Rev. Genet. 9:699–712.
24. Kashima, I., et al. 2006. Binding of a novel SMG-1-Upf1-eRF1-eRF3 com-
plex (SURF) to the exon junction complex triggers Upf1 phosphorylation
and nonsense-mediated mRNA decay. Genes Dev. 20:355–367.
25. Kaygun, H., and W. F. Marzluff. 2005. Regulated degradation of replication-
dependent histone mRNAs requires both ATR and Upf1. Nat. Struct. Mol.
Biol. 12:794–800.
26. Kaygun, H., and W. F. Marzluff. 2005. Translation termination is involved in
histone mRNA degradation when DNA replication is inhibited. Mol. Cell.
Biol. 25:6879–6888.
27. Kedersha, N., and P. Anderson. 2002. Stress granules: sites of mRNA triage
that regulate mRNA stability and translatability. Biochem. Soc. Trans. 30(Pt.
28. Kedersha, N., and P. Anderson. 2009. Regulation of translation by stress
granules and processing bodies. Prog. Mol. Biol. Transl. Sci. 90:155–185.
29. Kedersha, N., S. Chen, N. Gilks, W. Li, I. J. Miller, J. Stahl, and P. Ander-
son. 2002. Evidence that ternary complex (eIF2-GTP-tRNAi
preinitiation complexes are core constituents of mammalian stress granules.
Mol. Biol. Cell 13:195–210.
30. Kedersha, N., et al. 2000. Dynamic shuttling of TIA-1 accompanies the
recruitment of mRNA to mammalian stress granules. J. Cell Biol. 151:1257–
31. Kedersha, N., et al. 2005. Stress granules and processing bodies are dynam-
ically linked sites of mRNP remodeling. J. Cell Biol. 169:871–884.
32. Kedersha, N. L., M. Gupta, W. Li, I. Miller, and P. Anderson. 1999. RNA-
binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2? to the
assembly of mammalian stress granules. J. Cell Biol. 147:1431–1442.
33. Kedersha, N. L., and L. H. Rome. 1986. Isolation and characterization of a
novel ribonucleoprotein particle: large structures contain a single species of
small RNA. J. Cell Biol. 103:699–709.
34. Kim, W. J., S. H. Back, V. Kim, I. Ryu, and S. K. Jang. 2005. Sequestration
of TRAF2 into stress granules interrupts tumor necrosis factor signaling
under stress conditions. Mol. Cell. Biol. 25:2450–2462.
35. Kozlov, S., N. Gueven, K. Keating, J. Ramsay, and M. F. Lavin. 2003. ATP
activates ataxia-telangiectasia mutated (ATM) in vitro: importance of auto-
phosphorylation. J. Biol. Chem. 278:9309–9317.
36. Li, W., M. Simarro, N. Kedersha, and P. Anderson. 2004. FAST is a survival
protein that senses mitochondrial stress and modulates TIA-1-regulated
changes in protein expression. Mol. Cell. Biol. 24:10718–10732.
37. Liu, S. X., et al. 2005. Mitochondrial damage mediates genotoxicity of arse-
nic in mammalian cells. Cancer Res. 65:3236–3242.
38. Masse, I., et al. 2008. A novel role for the SMG-1 kinase in life span and
oxidative stress resistance in Caenorhabditis elegans. PLoS One 3:e3354.
39. McIlwain, D. R., et al. 2010. Smg1 is required for embryogenesis and regu-
lates diverse genes via alternative splicing coupled to nonsense-mediated
mRNA decay. Proc. Natl. Acad. Sci. U. S. A. 107:12186–12191.
40. Medghalchi, S. M., et al. 2001. Rent1, a trans-effector of nonsense-mediated
mRNA decay, is essential for mammalian embryonic viability. Hum. Mol.
Genet. 10:99–105.
41. Meslin, F., et al. 2011. hSMG-1 is a granzyme B-associated stress-responsive
protein kinase. J. Mol. Med. 89:411–421.
42. Morita, T., et al. 2007. Distant N- and C-terminal domains are required for
intrinsic kinase activity of SMG-1, a critical component of nonsense-medi-
ated mRNA decay. J. Biol. Chem. 282:7799–7808.
43. Muller, B., J. Blackburn, C. Feijoo, X. Zhao, and C. Smythe. 2007. DNA-
activated protein kinase functions in a newly observed S phase checkpoint
that links histone mRNA abundance with DNA replication. J. Cell Biol.
44. Nadezhdina, E. S., A. J. Lomakin, A. A. Shpilman, E. M. Chudinova, and
P. A. Ivanov. 2010. Microtubules govern stress granule mobility and dynam-
ics. Biochim. Biophys. Acta 1803:361–371.
45. Ohn, T., N. Kedersha, T. Hickman, S. Tisdale, and P. Anderson. 2008. A
functional RNAi screen links O-GlcNAc modification of ribosomal proteins
to stress granule and processing body assembly. Nat. Cell Biol. 10:1224–1231.
46. Oliveira, V., et al. 2008. A protective role for the human SMG-1 kinase
against tumor necrosis factor-alpha-induced apoptosis. J. Biol. Chem. 283:
47. Roberts, T. L., J. A. Dunn, M. J. Sweet, D. A. Hume, and K. J. Stacey. 2011.
The immunostimulatory activity of phosphorothioate CpG oligonucleotides
is affected by distal sequence changes. Mol. Immunol. 48:1027–1034.
48. Sarkaria, J. N., et al. 1998. Inhibition of phosphoinositide 3-kinase related
kinases by the radiosensitizing agent wortmannin. Cancer Res. 58:4375–
49. Sheth, U., and R. Parker. 2006. Targeting of aberrant mRNAs to cytoplasmic
processing bodies. Cell 125:1095–1109.
50. Shevchenko, A., M. Wilm, O. Vorm, and M. Mann. 1996. Mass spectrometric
sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68:
51. Shor, B., J. J. Gibbons, R. T. Abraham, and K. Yu. 2009. Targeting mTOR
globally in cancer: thinking beyond rapamycin. Cell Cycle 8:3831–3837.
52. Solomon, S., et al. 2007. Distinct structural features of caprin-1 mediate its
interaction with G3BP-1 and its induction of phosphorylation of eukaryotic
translation initiation factor 2?, entry to cytoplasmic stress granules, and
selective interaction with a subset of mRNAs. Mol. Cell. Biol. 27:2324–2342.
53. Stephens, P., et al. 2005. A screen of the complete protein kinase gene family
identifies diverse patterns of somatic mutations in human breast cancer. Nat.
Genet. 37:590–592.
54. Thomas, M. G., M. Loschi, M. A. Desbats, and G. L. Boccaccio. 2011. RNA
granules: the good, the bad, and the ugly. Cell Signal. 23:324–334.
55. Wang, D., et al. 2011. Inhibition of nonsense-mediated RNA decay by the
tumor microenvironment promotes tumorigenesis Mol. Cell. Biol. 31:3670–
56. Weischenfeldt, J., et al. 2008. NMD is essential for hematopoietic stem and
progenitor cells and for eliminating by-products of programmed DNA rear-
rangements. Genes Dev. 22:1381–1396.
57. Wong, M. P., et al. 2003. Chromosomal aberrations of primary lung adeno-
carcinomas in nonsmokers. Cancer 97:1263–1270.
58. Yamashita, A., et al. 2009. SMG-8 and SMG-9, two novel subunits of the
SMG-1 complex, regulate remodeling of the mRNA surveillance complex
during nonsense-mediated mRNA decay. Genes Dev. 23:1091–1105.
59. Yamashita, A., T. Ohnishi, I. Kashima, Y. Taya, and S. Ohno. 2001. Human
SMG-1, a novel phosphatidylinositol 3-kinase-related protein kinase, asso-
ciates with components of the mRNA surveillance complex and is involved
in the regulation of nonsense-mediated mRNA decay. Genes Dev. 15:2215–
VOL. 31, 2011 ROLE FOR hSMG-1 IN STRESS GRANULE FORMATION 4429


text:
Reader comments are listed below. Comments are currently closed and new comments are no longer being accepted.
"Pakistan will have to live with the jihadists it promotes"
Is the Economist serious? THE CIA CREATED AND FUNDED THE HAQQANI NETWORK. This latest smear campaign against Pakistan will only backfire. Mullen's unsubstantiated allegations have only unified the country and have managed to unite all of the country's variegated political parties. You reap what you sow. The CIA actively promoted militant Islam during the 1980s, and now their brainchild has turned into their worst enemy.
Pakistan has lost more than 30,000 civilians in the "war on terror" and more than 6,000 soldiers. No wonder why the readership of the Economist is decreasing, as people turn to more intelligent analytical viewpoints from The Huffington Post and al Jazeera.
I can see how this is a tough spot for Pakistan because of their fear of an attack from their neighbors, but giving the Haqqani group the inch they need to "help out" the government may later lead to a mile. Some may say they are taking the lesser of two evils (from already a bad situation), but turning a blind eye to the location and attacks of the Haqqani network is just cultivating future problems.
I'm most disturbed about the statement made in the beginning of this article about Pakistani militia throwing their "crazies" into Afghanistan. Though the officer is retired, I think his brutal insight into the stance Pakistan has taken is both true and troubling. It also aligns with what American officials have laid claim to.
The Economist explains: Why the euro zone is recovering
The Economist explains
The Economist explains: How does a denial-of-service attack work?
The Economist explains
Prada's bad year: Fashion disaster
Business and finance
Saudi Arabia and Yemen: An easier road in than out
Middle East and Africa
Nigeria's election: Hail to democracy
Middle East and Africa
Yemen: The old man who won't go away
Middle East and Africa
Britain's election: Not keeping it real
Have you listened to The Economist Radio on Facebook?
The Economist Radio is an on-demand social listening platform that allows you to listen, share and recommend The Economist audio content
Test your EQ
Take our weekly news quiz to stay on top of the headlines
In Other Words
Try our new audio app and website, providing reporting and analysis from our correspondents around the world every weekday
Want more from The Economist?
Visit The Economist e-store and you'll find a range of carefully selected products for business and pleasure, Economist books and diaries, and much more

subset: commoncrawl


text:
Three questions--meristem--yellow AV
Have you tried meristem propagation? It sounds interesting, if technical.
Linda, Irina, Pembroke,
Has anyone tried any of the new yellow AVs? Just curious, since we have been talking about white AVs.
The third question
is about sending leaves, whether it is best to send a freshly cut leaf, or a leaf that has been started and has a few tiny roots?
This post was edited by fortyseven on Fri, Mar 7, 14 at 23:08
Meristem propagation is generally only used for plants that are difficult or impossible to propagate in any other way. It is commonly used for orchid growing. It is interesting. It is also time-consuming, requires special lab equipment and must be done in a sterile environment. It also takes a lot of practice to be successful. When I was in school we did a meristem project and all anyone got was a flask full of mold!
With African violets being so easy to propagate, there is no need to resort to this means of propagation. Break off a leaf, stick it in a pot and wait. How easy is that!
I admit it was fun to try meristeming but no one really has access to the equipment needed to do so.
Send your leaves freshly cut. It used to be the tradition to send them with a bit of moist cotton or paper toweling at the end but this has fallen out of favor as it can cause the leaf to rot if too wet for too long. However, leaves were sent this way for years, so I guess whatever works, works.
I would not send a rooting leaf unless you had no other options. The leaf is attempting to push out roots and I would think something as disrupting as boxing them up and giving them to the post office to abuse for days would not be a good idea. If I had to send a rooted leaf, I would either pot it up or wrap the end with moist cotton.
My thoughts on the 'yellow' violets. I'm a purist when it comes to plants. As a horticulturist, I know a plant can only produce the colors for which it has the pigments. Violets do not have the pigments for yellow so what we are seeing is a light green. I know I am being picky but I can't look at a yellowish violet without thinking, "This doesn't look right!" (I do this with all plants. I can't even look at the wildly dyed bouquets in Kroger! And yellow-leaved plants!!! In my head yellow leaves =dying plant!)
I've included a link to a discussion of "yellow" violets ...
I would suggest that you try "sunkissed rose".
Though it was essentially, a white/yellow/rose blend, sometimes it shows quite a bit of yellow.
Also, it seems to be a good base for propagating "more yellow" violets, per the linked discussion,
The plant is also quite vigorous and easy to propagate.
Here is a link that might be useful: Violet Voice discussion on Yellow African Violets
This post was edited by aegis500 on Tue, Mar 11, 14 at 21:52
Aegis, this sport is very yellow! Although I like it, had to agree with Linda, it looks odd. I have some issues with colors on my own; blue and and highlighter shades aren't edible for me. If there isn't real edible coming in the color, there shouldn't be even frosting in that shade! If you guessed that I don't eat blue mm candies you are right!
Meristem propagation is pain, I would stick to leaves, most of them eventually grow babies with no effort from our side ;)
Hi Aegis,
Thanks so much for the beautiful photos, the link, and especially the advice from growing it yourself. I have been researching yellows a little, and this was my first choice. So reading of your experience confirms it.
This post was edited by fortyseven on Mon, Mar 10, 14 at 23:40
Joanne -
I think Linda covered the issue.
I was at the lecture on meristem propagation - and as a handout - I got a test tube of Kohleria starters, Grew them, separated - and shared.
There are issues - it should be done in lab conditions - otherwise they will rot, why do I need 200 starters on the same plant, and - for whatever reason the M-propagation shakes the stability of the plant - and AV usually give a very high percent of mutations. So you have 200 starters - and they are not blooming true...
Linda & Irina,
I think I would have enjoyed being in the science lab with you trying meristem. Although Irina, I do agree, 200 starters would be a lot.
The photos of the yellow violets are absolutely amazing.
I plant to get a few yellows and will let you know.
Yellow is a great color in nature. The eye just needs to adjust.
This post was edited by fortyseven on Tue, Mar 11, 14 at 23:42
It was very interesting-the equipment and techniques used. I also have a friend of a friend who has a company propagating orchids by tissue culture. We visited her place when we were in LA on our way to Tokyo. She showed me the whole process and showed me the end results-she was actually getting orchids and not flasks full of mold!
Orchids still seem advanced level for me. I am not sure why, as they are low maintenance.
They are very common here for ornaments in every kind of store or hotel or beauty shop or restaurant because the natural weather is conducive. They are also very tidy looking and neat, no messy soil to spill. People use them to decorate instead of cut flowers. At parties, for table decorations, people can take them home. People who
use them in their shops for ornament just rotate them out.
Once they stop blooming, they put them into their "orchid farm," usually, their back porch where they are retired for a while.
They seem to consider it a bonus if they bloom again.
A friend of mine has a green thumb for orchids but not
violets. I had to take care of them for her. I would give her my dormant orchids, she would get them to bloom.
It is probably just a matter of learning what they need and not expecting too much from them after the first bloom cycle (orchids).
This post was edited by fortyseven on Tue, Mar 18, 14 at 1:34
A lot of people in your area grow Odontoglossums and Oncidiums. Miltoniopsis also. They do well in your climate and are so pretty.
It's too hot here for them without a greenhouse but my Paphs and Phrags seem to love the crazy weather!
The trick to success is to grow what suits your climate and try not to yearn after the ones that don't. Easier said then done; Miltoniopsis are my favorite! ;)


text:
City Guides: 2 Centuries of SF Architecture | Downtown
Dang! This event has already taken place.Sunday, April 22, 2012 - 11:00 am | Cost: FREE
>> Want to see our Top Picks for this week instead?
>> Want to see our Top Picks for this week instead?
| Stockton and Maiden Lane, San Francisco, CA
Explore downtown architecture from as early as 1891 up to today. You'll see famous buildings and little-known treasures while discussing the architects and design trends that changed the face of America. 30 buildings in two hours.
These free, regularly scheduled tours are offered only for individuals and groups of 7 or fewer persons. Groups of more than 7 must arrange for a private group tour.
Links: Event detailsCost: FREE
Free Stuff, Lectures & Workshops, San Francisco
Address: Stockton and Maiden Lane, San Francisco, CA
Submitted by Kelsey Bryant


text:
D. Marvin Jones
Attorney Misconduct, Drinking, Insider Trading, Law Schools, Legal Ethics, Morning Docket, Prostitution, Sentencing Law
* Three days after arguing that an alleged Sandusky victim's lawsuit lacked any factual basis, Second Mile decided to settle. Better strike while the iron is hot (and the wallet is open), lawyers. [Bloomberg] * So much for that "real shot," huh? After a failed bid for bail, Galleon Group's Raj Rajaratnam will begin serving […]
5th Circuit, Benchslaps, Contests, D. Marvin Jones, Edith Jones, Law Professors, Lawyer of the Day, Murder, Prostitution, Reader Polls, Sex, Sex Scandals, Wall Street
Lawyer of the Month: September Reader Poll
Our candidates for the coveted Lawyer of the Month title have been a bit tame for the past few months. This time around, we've chosen some lawyers and law students who represent our more prurient interests and our unabashed love for scandal. Aficionados of hookers? We've got 'em. Vicious tongue lashings? We've got those, too. […]
What Do Lawyers And Celebrities Have In Common?
Besides their good looks and fame, they're also increasing their focus on data security. In the wake of "Celebgate," the Sony Pictures hack, and nearly daily data breaches targeting massive corporations to individuals, law firms are finally recognizing the importance of bringing their cybersecurity policies up to speed.
Crime, D. Marvin Jones, Law Professors, Law Schools, Prostitution, Quote of the Day, Sex, Sex Scandals
Quote of the Day: Driving a Mercedes But Seeking a Hummer?
I am just a horny guy. – a comment allegedly made to the police by University of Miami law professor D. Marvin Jones, upon being arrested for a prostitution-related offense last month. (This is not the first time Professor Jones has been accused of such a crime. Back in 2007, we named him a Lawyer […]
Crime, D. Marvin Jones, Law Professors, Law Schools, Prostitution, Sex, Sex Scandals
Miami Criminal Law Professor Accused of Prostitution-Related Offense — Again
In October 2007, Professor D. Marvin Jones of the University of Miami School of Law was hit with a misdemeanor charge of soliciting a prostitute. We found it interesting that a criminal procedure professor was charged with, well, violating the criminal law. We bestowed Lawyer of the Day honors upon Professor Jones. Well, it seems that Professor Jones is back. Multiple University of Miami law school sources have alerted us to a new charge against him….
Admin, Announcements, Blogging, D. Marvin Jones, Defamation, Free Speech, Law Professors, Lawsuit of the Day, Media and Journalism, Pro Se Litigants
Breaking: Jones v. Minkin Dismissed!!!
(Plaintiff voluntarily dismisses lawsuit against ATL.)
We've written a fair amount about D. Marvin Jones, the University of Miami law professor who has been accused of soliciting an undercover officer for sex. He allegedly offered her a tantalizing $20 for her services. But a picture is worth a thousand words. And a picture is what's been making the rounds among UM […]
D. Marvin Jones, Law Professors, Law School Deans, Law Schools, Prostitution
As we first reported yesterday,
Title: ACA 5 Year Anniversary – Top 5 Issues in Medicare & Medicaid
A goal of the Patient Protection and Affordable Care Act is to provide quality, affordable healthcare for all Americans. Now in its 5th year, how much progress has been made in Medicare and Medicaid? Download Wolter's Kluwer's Special Report Here.
Crime, D. Marvin Jones, Law Professors, Lawyer of the Day, Prostitution, Sex Scandals
Lawyer of the Day: D. Marvin Jones
Several commenters to our recent post about the University of Miami law student who got benchslapped on the People's Court pointed out another news development involving the law school: the recent arrest and arraignment of a popular professor, D. Marvin Jones, on a misdemeanor charge of soliciting a prostitute. See here: Check out his bio […]


text:
Showing all newswire headlinesView by date, instead?
« Previous ( 1 ... 4235 4236 4237 4238 4239 4240 4241 4242 4243 4244 4245 ... 4730 ) Next »
Quasar Accounting finds move to open source adds up
Last month Linux Canada released Quasar Accounting 1.4 for Linux under the terms of the GNU General Public License. Phil Tonnellier, president of Linux Canada, says it's too early to tell if the move to open source is paying off, but response has been good so far, with more than 6,000 downloads since the January 13 release.
New P2P Internet Audio Idea
Imagine combining BitTorrent and streaming audio. Instead of a web host and mirrors serving up streaming audio content at their expense, each listener of the audio becomes a potential server for it.
Gentoo Weekly Newsletter -- February 28, 2005
A fabulous first European Gentoo developer meeting and the bustling atmosphere of the FOSDEM conference in Brussels last Saturday and Sunday, news from Apache and documentation for Gentoo/FreeBSD: The Gentoo Weekly Newsletter is late this week, but hopefully worth the wait. Gentoo in the press still contains indirect fallout from the Boston LWE, and an interesting article about Gentoo in the enterprise, and the community section introduces a new mailing list archive for catalyst developers and users. Our usual GLSAs and bug statistics complete the picture for this week, enjoy reading the GWN!
Report of Paris Solutions Linux 2005
Solutions Linux trade show is the French annual rendez-vous of Free Software technologies and their commercial applications. This year, it ran from February 1st to February 3rd. Like preceding years, KDE-France was present and benefited of a free booth in the "Associative Village".
Resurgent Novell: Building Linux for Grownups?
My heart's desire, as a gnarly old Linux/Windows sysadmin, has long been for better management utilities: directory services, user and resource management, system monitoring, and single sign-on that work across a mixed environment. Something like Active Directory, only without the vendor lock-in, and it works right....In a sane world, Novell NetWare would have remained the dominant network operating system.
How secure is your computer?
"Honey pot" experiment shows unprotected Windows SP 1 at risk
First look: OpenOffice.org version 2.0 beta
OpenOffice.org has always been conservative with version numbers. Enough minor releases have boasted enough new features that the current release could easily be 3.0 or 4.0 instead of 1.1.4. Given this record, it's hardly surprising that version 2.0, for which beta code was set to be unveiled yesterday, amounts to a major rewrite of the software. Although key functionality remains largely intact, version 2.0 promises dozens, possibly hundreds, of changes. Many times during our testing of the first beta release, we felt we could almost have been looking at an entirely new piece of software.
Basic Tasks for New Linux Developers
Linux systems require you to log in, become the super user (or root) for some tasks, open a terminal or shell window, and mount a CD-ROM. If you are new to Linux, this article will guide you through these tasks and more.
Quantum GIS : Interview with Marco Hugentobler
This is the fifth in our series of interviews with QGIS developers and users. This week we travel to Zurich, Switzerland to chat with QGIS developer Marco Hugentobler. Marco works on the vector data model implementation in QGIS. The interview was conducted by Tim Sutton and Gary Sherman.
New FSF Europe fellowship program announced
The Free Software Foundation Europe has announced a new Fellowship program to defend freedom in the digital age. Loosely modeled on the US-based Free Software Foundation's Associate Membership program, the FSFE hope to attract both more finances and activists to support their work. At a time when the free software movement is under a variety of threats, both legal, political and market-based, the FSFE hope that its Fellowship will be seen as "a call to arms."
Debian Project Leader Election 2005
The nomination period is at an end, with six candidates standing forth to be counted. We are now in the campaigning period.
The LiveCD List Updated
Since last year's release, The LiveCD List has more than doubled to a total of 220 LiveCDs. Today an updated version of The LiveCD List is being released on its own domain name, LiveCDList.com. It includes not only Linux x86 and PPC LiveCDs, but also 9 other architectures, BSD, Windows, LiveDVDs, and it links directly to the projects' download pages.
Pie Box Enterprise Linux 4 AS Launched
PixExcel today released version 4 of its Pie Box Enterprise Linux product. Pie Box Enterprise Linux 4 AS is built from the source RPMs of Red Hat Enterprise Linux 4 AS. It is fully compatible with Red Hat Enterprise Linux 4 and features the Linux 2.6 kernel, SELinux, GNOME 2.8, Samba 3.0, Logical Volume Manager 2, PCI Express support and NFSv.4.
KDE 3.4 Release Candidate 1
On February 26th 2005, the KDE Project announced the first release candidate of KDE 3.4. Compile the sources (KDE 3.4 requirements list, "Konstruct" build script), download the "Klax" i486 GNU/Linux Live-CD (375 MB) or the first contributed binary packages. More packages may follow later. Please test the new features and report all bugs so that we can identify the show-stoppers to be fixed before the final release planned for 16th March. OSdir.com is the first to have screenshots of KDE 3.4 RC 1 and tuxmachines.org shows how customizable its look is.
Linux: Improving Multiprocessor CPU Scheduling
Nick Piggin uploaded a series of patches for the 2.6 Linux kernel CPU scheduler aimed at improving multiprocessor support. Specifically, the patches focus on improving SMT (Symmetric MultiThreading), CMP (Cellular MultiProcessing), and NUMA (Non-Uniform Memory Architecture) scheduling behavior.
Timing release schedules to maximize publicity
Two or three times each year, Macintosh enthusiasts go nuts over Apple's new product launches, and the company rides a wave of publicity from the resulting media attention. Open source projects would do well to study how Apple orchestrates this, because publicity is the key to attracting new users -- and new users are the lifeblood of the development model.
Gentoo Linux Is Coming into Its Own
Too raw for enterprise use now, but the distribution's worth watching.
Microsoft's C++ compiler has violated the ISO language standard for at least the last three versions
...I think that Microsoft's C++ compiler has violated the ISO language standard at least for the last three versions, and they are now fixing the bug in their C# compiler. Think of all that MS code out there that is broken!
UFT Gives Lesson in Open Source
Teachers' union trades paper forms for Web services and online transactions.
Linux Isn't for EveryoneYet
The drive to Linux and other open-source platforms presents a host of benefits.
« Previous ( 1 ... 4235 4236 4237 4238 4239 4240 4241 4242 4243 4244 4245 ... 4730 ) Next »


text:
1 of 1 people found the following review helpful
Noise, Pure Unadulterated Noise from LA,
This review is from: Erect the Youth Problem (Audio CD)
Wives were just pure underground fuzzy noise that put many "hardcore" bands to shame. There's no screaming involved, but the over-distorted squealing guitar more than compensates for the drowned out ramblings.
The album is made up of break-neck speed 80's hardcore style songs that collapse just shy of a blink of an eye. The drumming is what holds these songs together. Though it's short, it does start to get samey towards the end.
Interesting fact...the money used to make this record came from a Backstreet Boy's insurance payment (he smashed into the singer/guitarist's car). Shame then (as you've noticed the past tense) that this band lasted a just under a year before packing it. Check this album out if you love that old skool DC hardcore noise.
Review Details
(2 customer reviews)
Location: Cardiff, Wales UK
Top Reviewer Ranking: 6,061,842


text:
Posted| Updated
At least 13 people were killed on Thursday in an air assault by US forces on a house in Iraq's restive city of Ramadi, according to medics.
Topics: unrest-conflict-and-war, iraq, united-states
Posted| Updated
There has been a double road fatality near Ipswich in south-east Queensland.
Topics: disasters-and-accidents, ipswich-4305
Posted| Updated
Australia and Singapore will lead an international exercise in June to test communications preparedness for a possible bird flu pandemic, officials said on Thursday.
Topics: emergency-planning, australia, singapore
Posted| Updated
Hundreds of people were injured as thousands of South Korean riot police fought pitched battles on Thursday with anti-US protesters and villagers as the military started clearing a site for a new US army base.
Topics: unrest-conflict-and-war, korea-republic-of, united-states
Posted| Updated
Blackburn manager Mark Hughes has agreed a new contract that will keep him at the English Premiership club until at least 2009.
Topics: english-premier, england
The only man charged in the United States over the September 11 terrorist attacks will spend the rest of his life behind bars in the highest security prison in America.
Topics: courts-and-trials, terrorism, united-states
20060504-closer-pm - abc_news
The opposition candidate Manasseh Sogavare has been chosen as the new Prime Minister of Solomon Islands.
Topics: elections, solomon-islands
Posted| Updated
The United States has hit back at allegations that the CIA has flown thousands of planes with suspects aboard through Europe to third countries where they might face torture.
Topics: terrorism, united-states, european-union
Posted| Updated
Civil defence authorities in New Zealand are assessing emergency procedures, after an overnight tsunami alert triggered panic among residents of a coastal community on the North Island.
Topics: earthquake, new-zealand, tonga
Posted| Updated
The AFL Commission met with the 16 clubs on Thursday to discuss a proposed funding boost for the next five years.
Topics: australian-football-league, australia
The Federal Opposition has attacked the Treasurer over a newspaper photograph of him reportedly jumping for joy on the same day the Reserve Bank lifted interest rates.
Topics: consumer-finance, corporate-governance, economic-trends, regulation, small-business, australia
Palestinian President Mahmoud Abbas says he intends to ask Palestinians to vote by referendum on any peace deal with Israel, sidestepping the Hamas-led Government.
Topics: world-politics, unrest-conflict-and-war, palestinian-territory-occupied, israel
The High Court has begun hearing a challenge to the industrial relations reforms initiated by the Federal Government.
Topics: industrial-relations, courts-and-trials, australia
The Western Australian Government has refused to guarantee the future of the state's Science Council.
Topics: states-and-territories, science-and-technology, wa
A Sydney court has granted bail to the man accused of killing Sydney model Caroline Byrne.
Topics: courts-and-trials, nsw
A radical plan to overhaul taxes and services in Queensland has met with a resounding "no" from the Queensland Government.
Topics: states-and-territories, business-economics-and-finance, qld
Posted| Updated
The vast looping system of air currents that fuels Pacific trade winds and climate from South America to South-East Asia may be another victim of climate change, according to scientists.
Topics: climate-change, research, asia, australia, united-states
Two trapped Tasmanian miners are facing their 10th night underground as the rescue operation enters its most dangerous phase.
Topics: mining-industry, disasters-and-accidents, tas
Two trapped Tasmanian miners are facing their 10th night underground as the rescue operation enters its most dangerous phase.
Posted| Updated
The two trapped Tasmanian miners are spending their 10th night underground as the rescue operation enters its most dangerous phase.
Topics: emergency-incidents, mining-industry, beaconsfield-7270, launceston-7250
South Australian fast bowler Shaun Tait says he will be using Australia A's upcoming matches against New Zealand, India and Pakistan in Darwin and Cairns to push for a position in the Test line-up for the Ashes series later this year.
Posted| Updated
South Australian fast bowler Shaun Tait says he will be using Australia A's upcoming matches against New Zealand, India and Pakistan in Darwin and Cairns to push for a position in the Test line-up for the Ashes series later this year.
Posted| Updated
A study has found the guidelines used in the treatment of asthma are out of date.
Queensland utility back Drew Mitchell is expected to sign a three-year deal with the Western Force next week.
Topics: super-12, brisbane-4000, perth-6000


text:
'); } -->
2 bed, 2 full bath. One Level Home. Beautifully cared for with gleaming Hardwoods. Shows like a dream. Private locationMore info
2 bed, 1 full bath, 1 half bath. Vacation at home in this condo on Lake Murray. 2 BR 1. 5 Bath and private patio for entertaining. BMore info
2 bed, 1 full bath. Charming condo features hardwood floors, updated kitchen granite & stainless appliances. 9' smoothMore info
2 bed, 1 full bath. Gorgeous remodeled condo, updated in 2008 with granite counter tops,stainless steel appliances,refiMore info
2 bed, 1 full bath, 1 half bath. FHA Approved Condominium Neighborhood on Gorgeous Lake Murray! Seller To Pay $2500 of Buyer ClosingMore info
2 bed, 2 full bath. Exceptional 2 bedroom 2 bath condo located in the center of the harbison community. This wonderfulMore info
2 bed, 2 full bath, 1 half bath. Don't miss this updated wonder located on Carys Lake. Contemporary lakefront living. There are beauMore info
3 bed, 2 full bath. Super Patio home in Twin Oaks of Reflections, located on the end of a Cul-de-Sac offers 3BR/2BA, anMore info
2 bed, 2 full bath. WOW factor!! New neutral carpet/paint. Custom drapes. New dishwasher & kitchen hardware. Absolute mMore info
2 bed, 2 full bath. Spacious 2 bedroom/2 full-bath condo on lower-level in Rosewood. Turn-key with all kitchen and launMore info


text:
Country/Date of origin: France/1700s
The small sheepdog of the Pyrenees Mountains is considered the oldest of the French herding dogs. They were used in concert with the large white sheep guardian dogs of the region. The little dog herded the sheep. The big dog watched over them. The natural abilities of this shaggy shepherd are legend - flocks being entrusted to the two different dogs without a human handler. They were officially recognized by the French Kennel Club in 1921 but it has not made much of a name for itself outside its homeland.
FCI Group: 1 (Herding)
About This Breed:
Other Names
Body Type
Health Concerns
Petmate Sky Kennel Pet Carrier
Starting at: $48.99
Herm Sprenger Prong Collar
Starting at: $15.99
Frontline Plus for Dogs 6 Mths
Starting at: $79.19
Greenies Dog Dental Chew Treats Regular
Starting at: $13.59
MidWest Solutions 2 Door Large Dog Crate
Now: $147.99
Greenies Dog Dental Chew Treats Large
K9 Advantix II for Dogs 6-Month Supply
Starting at: $63.99
Greenies Dog Dental Chew Treats Teenie
Petite Greenies Dog Dental Chew Treats
Slip Lead for Dogs
Starting from: $8.99
Midwest Puppy Playpen w/ 1/2" Grid
Starting from: $128.99
MidWest Life-Stages Folding Dog Crate
Starting from: $29.99
LitterMaid Classic Mega Multi-Cat Litter Box
Now: $139.99
MidWest Pet Exercise Pen
Starting from: $38.99
Wahl Arco SE Continuous Cordless Clipper
Starting at: $132.49
Midwest Life-Stages Double Door Dog Crate
Starting at: $32.99
Pet n Shape Chik n Skewers Dog Treat
Starting at: $19.49
Frontline Plus for Dogs 3 Mths
Starting at: $41.99
MidWest iCrate Dbl Door Folding Dog Crate
Starting from: $28.99
Pig Ears
Starting at: $33.99
Sometimes, manufacturers require that products be advertised only at their suggested prices.
When we choose to sell these items for less, we display the sale price only in the cart. To see the price, click Add to Cart.
Labri, Pyrenean Sheepdog
AKC Group: Miscellaneous
Breed Club:
Rescue Club:
Body Type:
Health Concerns:


text:
From baby animals to wild creatures to exotic pets, discover cute pictures and more about animals.
About StumbleDNA StumbleDNA
sobonsar's Interests
Whether you like romantic comedies or black comedies, have a laugh by Stumbling through funny films.
Get ideas for fun and easy DIY arts and crafts perfect for kids, holidays and more. Do it yourself!
Ready to get your groove on? Explore the best dance music, songs, videos, lyrics, bands and more.
Explore dancing, from ballroom to salsa, and soon you'll be wow-ing everyone with your moves.
Explore the art of design, from graphic design on the Web to interior design in the home.
Learn more about man's best friend: big or small, mutts or purebreds, puppies or old-timers.
Do you dream of traveling through Italy or France? Learn more about Europe (and plan your next vacation).
Are you a foodie? Expand your culinary horizons with new recipes, cooking ideas and nutrition tips.
Funny jokes, pictures or quotes...whatever your sense of humor, we have something to make you laugh.
How'd they do that!? Channel your inner wizard with card tricks, optical illusions, spells and more.
Are two wheels better than four? Whether you love Hondas, Yamahas or BMWs, explore motorcycles here.
Always first in line for the best new movies? Explore the magic of Hollywood and the cinema.
Music makes the world go round. Discover new bands, songs, lyrics, music videos and more.
Explore the wonders of Mother Nature and the science of earth's resources, climates and wildlife.
Whether in NYC, Miami, Vegas or LA, find out about the hottest lounges, bars or clubs.
It's been said that pets help you live longer. Sounds good to us! Learn about dogs, cats, fish and more.
See amazing photos and get photography tips and ideas, from Polaroids to digital cameras.
The human brain is like a muscle. Strengthen it with crosswords, jigsaw puzzles and word games.
Get your quote-of-the-day fix with funny sayings, famous proverbs and nuggets of inspiration.


text:
(September 15, 2011: O'Neill, NE 68763) The Wagner Red Raiders volleyball team scored 3 points and limited the host O'Neill Eagles to 1 in the Red Raiders non-league victory on Thursday.
The Red Raiders now possess a 7-2 record. They put it on the line next when they travel to Parker for a non-league game on Saturday, September 17. Wagner will meet a Pheasants team coming off a 3-2 non-league win over Alcester-Hudson (Alcester, SD). The Pheasants record now stands at 11-0.
The Eagles (5-3) will now prepare for their game against Valentine (Valentine, NE). The Badgers enter the non-league game with a 7-4 record. In their last game, Valentine defeated Bennett County (Martin, SD), 3-0, in a non-league game.
Not a Member?
Join MaxPreps now for free access to rankings for thousands of teams across all sports.


text:
Zilliox installed as Rotary district governor
Larry Zilliox, a member of the Alexandria Rotary Club, was installed as governor of District 5950 in a ceremony held July 8 in Alexandria.
Zilliox is just the second Alexandria Rotarian elected to this office; Joe Perino served as district governor in 1968. Zilliox is one of 533 district governors worldwide.
Are you a newspaper subscriber but you don't have a Digital Access account yet? https://secure.forumcomm.com/?publisher_ID=40&event=subscriber/lookup.
You will need your subscription account number and phone number. Not sure if you have an account? Email us at email@example.com and we can help you.