Linking t rna localization with activation

Cell Cycle 9:15, 3112-3118; August 1, 2010; © 2010 Landes Bioscience
Report
3112 Cell Cycle Volume 9 Issue 15
*Correspondence to: Mette Prætorius Ibba; Email: ibba.2@osu.edu
Submitted: 03/05/10; Revised: 05/19/10; Accepted: 05/28/10
Previously published online: www.landesbioscience.com/journals/cc/article/12525
DOI: 10.4161/cc.9.15.12525
Introduction
Autophagy (macroautophagy) has recently gained tremendous
attention due to its important role in initiation and propaga-
tion of human diseases such as cardiovascular diseases, heart
failure, neurodegeneration and cancer.1-6
Detailed understand-
ing of how autophagy can be modulated is a prerequisite for the
development of strategies to use this pathway as a therapeutic
target.
Autophagy is a catabolic process that allows cells to degrade
and recycle cytosolic constituents, such as long-lived proteins and
organelles, by lysosomal degradation.7
The degradation products
(nucleotides, amino acids, sugars and lipids) are shuttled from
the lysosome to the cytosol, where they are recycled as building
blocks for vital cellular processes that sustain cell growth, i.e.,
protein synthesis and ATP generation. Under favorable growth
conditions, the autophagic pathway operates at a low constitu-
tive (housekeeping) level to provide cytoplasmic quality control
through elimination of damaged organelles and misfolded pro-
tein aggregates, thereby ensuring cell homeostasis. During cel-
lular stress such as nutrient deprivation, this pathway is strongly
induced. Even though autophagy may provide a temporary sur-
vival pathway for metabolically-stressed cells, it may also promote
cell death if excessively upregulated.8-11
Cells respond to nutrient deprivation a variety of ways. In addition to global downregulation of cap-dependent protein
synthesis mediated by the GCN2 and mTORC1 signaling pathways, a catabolic process autophagy is upregulated to
provide internal building blocks and energy needed to sustain viability. It has recently been shown that during nutrient
deprivation tRNAs accumulate in the nucleus, but the functional role of this accumulation remains unknown. This study
investigates whether subcellular localization of tRNAs plays a role in signaling nutritional stress and autophagy. We report
that human fibroblasts that accumulate tRNA in the nucleus due to downregulation of their transportin, Xpo-t, show
reduced mTORC1 activity and upregulated autophagy. This suggests that subcellular localization of tRNAs may regulate
an intracellular response to starvation independently of the cellular nutritional status.
Linking tRNA localization with activation
of nutritional stress responses
Le-Nguyen Huynh,1
Muthusamy Thangavel,1
Timothy Chen,1
Ryan Cottrell,1
Joy Maria Mitchell1
and Mette Prætorius-Ibba1,2,
*
1
Department of Molecular and Cellular Biochemistry; College of Medicine; Comprehensive Cancer Center; 2
The Ohio State University; Columbus, OH USA
Keywords: tRNA, Xpo-t, mTOR, autophagy
Abbreviations: mTOR, mammalian target of rapamycin; GCN2, general control non-derepressible-2; tRNA, transfer RNA;
miRNA, microRNA; siRNA, small interfering RNA; Xpo-t, exportin-T; XPOT, gene encoding exportin-T; TNPO3, transportin
3; LC-3, MAPLC3B microtubule-associated protein 1 light chain 3B; GFP, green fluorescent protein; D. discoidium, Dictyostelium
discoideum; FITC, fluorescein isothiocyanate; DIG, digoxigenin; TEM, transmission electron microscopy; FISH, fluorescent in situ
hybridization; Kar, karyopherin; mTORC1, mammalian target of rapamycin complex 1
Regulation of the autophagic pathway is complex and involves
a number of different signaling cascades. Despite increasing
knowledge about these pathways, many key aspects remain
unknown. For example, whereas it is well-known that amino
acid depletion downregulates the activity of the serine/threonine
kinase mTOR (target of rapamycin) and upregulates autophagy
(Fig. 1), little is known about upstream factors which transduce
the starvation signal to mTOR. However, it was recently shown
that the four RAG GTPases make amino acid dependent interac-
tions with the mTOR kinase and thus play an important role in
amino acids sensing by mTORC1.12-14
We have observed that a yeast strain deleted for the tRNA
transporting karyopherin LOS1 showed increased autophago-
some-like punctates when expressing a GFP-tagged version of
the autophagic marker ATG8 (Fig. S1). This led us to sug-
gest that subcellular localization of tRNA may play a role in
autophagy regulation, perhaps serving as a sensor of amino
acid availability. To assess this hypothesis, and to extend
our analysis to the mammalian cells, we analyzed autophagy
regulation in human foreskin fibroblasts (HFF) that accu-
mulate tRNAs in the nucleus as a result of reduced expres-
sion of karyopherin Xpo-t. Our data demonstrate that Xpo-t
knocked down fibroblasts indeed show elevated autophagy.
Furthermore, the Xpo-t knocked down cells display reduced

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in the nucleus. The underlying reason for the latter is
yet unknown.26-28
To determine whether tRNA retention in the nuclei
of HFF would influence the autophagic process simi-
larly to the deletion of yeast LOS1 (Fig. S1), we chose
to knock-down the karyopherin Xpo-t by nucleofection
with XPOT-specific siRNA. Using both real-time PCR
and immunoblotting, we confirmed that the expression
of the karyopherin was reduced by more than 80% as
compared to cells nucleofected with a non-targeting
siRNA (Fig. 2A and B).
We next tested whether siRNA-mediated knock-
down of Xpo-t in fibroblasts would lead to tRNA
accumulation in the nucleus. Fluorescent in situ
hybridization (FISH) using a DIG-labeled oligonucle-
otide designed to hybridize to tRNALys
and fluorescent
anti-DIG antibodies, demonstrated that in control
cells, tRNALys
was accumulated in the nuclei only fol-
lowing starvation; this effect was reversed when the
cells were refed. By contrast, the Xpo-t knocked down
cells showed similar levels of increased nuclear accumu-
lation of tRNALys
under all conditions (Fig. 2C and D).
Nuclear and cytosolic extractions followed by quanti-
tative RT-PCR also confirmed an increase of tRNALys
(1.8 fold) in the nucleus in Xpo-t knocked down cells
as compared to control cells (Fig. 2E and F). These
experiments confirm that reduction in Xpo-t levels in
fibroblasts by siRNA knock-down impairs nucleocyto-
plasmic transport of tRNAs and that this effect is inde-
pendent of the cells nutritional status.
Knock-down of Xpo-t leads to changes of autophagic mark-
ers and accumulation of autophagosomes. To test whether
accumulation of tRNA caused by defects in nuclear export
contributes to the onset of a starvation-like response, the Xpo-t
knocked down fibroblasts were assessed for upregulation of
autophagy. We first examined changes in the expression level
and the modification state of the commonly used autopha-
gic marker LC3 (MAPLC3B microtubule-associated protein
1 light chain 3B), a mammalian ortholog of yeast Atg8. LC3
is a key component required for autophagosome formation in
mammalian cells: after LC3 is processed and conjugated to
phosphatidylethanolamine (PE), the lipidated (LC3-II) form
becomes tightly associated with the inner and outer autopha-
gosomal membranes. LC3-II plays an additional role in protein
targeting: it directly binds to, and selectively targets, p62 into
autophagosomes.29,30
LC3-I and its lipidated form, LC3-II, were visualized by
immunoblotting using protein extracts from HFF cells knocked
down for Xpo-t or treated with a non-targeting (control) siRNA
and siKar RNA directed towards TNPO3 karyopherin that has
not been shown to participate in tRNA export from the nucleus
(Fig. 3A). Extracts from HFF grown in nutrient replete media
were compared to those from cells grown in nutrient deplete
medium or medium containing chloroquine (CHL), which
blocks the later steps in autophagic pathway, such as lysosomal
degradation of LC3-II and p62.
activity of the mTORC1 pathway. These data support a role
for tRNAs in starvation signaling through their subcellular
localization.
Results and Discussion
siRNA-mediated knock-down of karyopherin Xpo-t in
human fibroblasts leads to nuclear accumulation of tRNA.
Nucleocytoplasmic trafficking of macromolecules such as
tRNAs is a tightly controlled process that relies on several fac-
tors including (1) karyopherins, transport receptors that recog-
nize, bind to, and assist in transport of, macromolecules across
the nuclear envelope; (2) nucleoporins that are confined to the
nuclear pore complex; and (3) RanGTPase that controls the
rate at which macromolecules are translocated.15
Xpo-t and its
yeast orthologue Los1 are tRNA-specific nuclear export recep-
tors; they bind newly synthesized tRNAs in the nucleus and
mediate their transport to the cytoplasm.16-18
Although los1 dele-
tion strains show impaired tRNA splicing and accumulation of
tRNAs in the nucleus, the LOS1 gene is not essential for growth,
suggesting that other, partially redundant tRNA transport sys-
tems exist.19,20
During amino acid deprivation, several cellular tRNA-related
events take place. One such event is the activation of the nutri-
ent-responsive kinase GCN2 by uncharged tRNAs present in the
cytosol.21-23,25
Another is the accumulation of cytoplasmic tRNA
Figure 1. The main mechanisms sensing nutrient availability. A summary of the
nutrient-responsive pathways and their role in autophagy regulation. Our hypoth-
esis that tRNA subcellular localization affects autophagy is also incorporated in the
figure. The circled components are of main interest to this study.

the observed change was not dramatic, we used an autophagic
marker p62/SQSTM to substantiate our findings. The p62/
SQSTM protein binds to and escorts proteins destined for lyso-
somal degradation to the autophagic vesicles. In the course of
this process, p62/SQSTM is itself sequestered and degraded in
the autolysosomes.29-31
Immunoblot analysis showed that in cells
grown in presence of nutrients the level of p62/SQSTM was
The lipidated form of LC3, LC3-II (which migrates as a
17 kD species) is easily distinguished from LC3-I (15 kD). As
expected, the levels of LC3-II were markedly increased in CHL-
treated samples (Fig. 3A). We observed a small yet reproducible
increase in LC3-II levels in CHL-treated cells knocked down
for Xpo-t (but not the cells treated with control and kar siR-
NAs) during both normal growth and during starvation. Since
Figure 2. Knock-down levels of Xpo-t and cellular localization of tRNALys
in HFF cells. HFF were transfected with a non-targeting control siRNA, with
siRNA towards Xpo-t or without any siRNA (none, mock transfected). Cells were grown in non-starvation or starvation medium (6 h); to test the
recovery from starvation, growth was continued in non-starvation media for 24 h following 6-h starvation. All individual siRNA transfections were
tested for knocked down levels of Xpo-t. (A) The Xpo-t mRNA levels were determined by quantitative RT-PCR and normalized to GAPDH mRNA. Error
bars indicate s.d.; **p < 0.01; n = 6. (B) Protein levels of Xpo-t measured by immunoblotting; actin was used as a control throughout this work. (C and
D) Subcellular localization of tRNAs in HFF cells was visualized by FISH using DIG-labeled probes complementary to human tRNACUU
Lys
and FITC-conju-
gated anti-DIG antibodies. Nuclei were visualized by DAPI staining. (E and F) Quantitative RT-PCRs were performed with nuclear or cytoplasmic RNA
isolated from luciferase or Xpo-t siRNA transfected HFFs. (E) 2.5% agarose gel loaded with the cDNA samples. (F) Relative levels of tRNALys
cDNA in the
cytoplasm or nucleus in the transfected cells were normalized to siRNA control levels.

Figure 3. Levels of mTOR and autophagic markers in XPO-T knocked down HFF cells. Levels of autophagic markers LC3 (A) and P62/SQSTM (B) were
determined by immunoblotting of extracts from HFF cells treated as described in Figure 2 legend. Where indicated, the cells were treated with
chloroquine (CHL) for 3 h. The levels of LC3-II and p62 (normalized to actin) are indicated below each lane. (C) Activity of the mTORC1 pathway was
assessed by measuring levels of phosphorylated forms of mTOR and S6 (mTORSer2448-P
and S6Ser235/236-P
, respectively) as compared to their total levels, as
well as those of a phosphorylated form of 4E-BP, 4E-BPThr37/46-P
, using immunoblotting. (D) Transmission electron microscopy was carried out to visual-
ize and quantify autophagosome structures (indicated by arrows) in cells nucleofected with a control siRNA or Xpo-t siRNA. (E) Autophagosomes were
counted and compared to the number of autophagosomes in non-starved cells treated with control siRNA.

We also probed the activity of another mTOR complex,
TORC2, which phosphorylates and activates AKT1 at position
Ser473 (AKTSer473-P
). We found no indication of altered AKT1
phosphorylation in cells knocked down for Xpo-t relative to con-
trol cells (Fig. S2). This suggests that the observed differences are
specific for the mTORC1pathway.
Xpo-t knock down increases autophagosome formation. One
of the most powerful ways to study upregulation of autophagy is
the direct visualization of the double-membrane autophagosomes
in the cytoplasm by transmission electron microscopy (TEM).
We found that after nutrient starvation for 24 hours, the cells
transfected with a control siRNA showed an ∼1.5 fold increase
in the number of autophagosomes (Fig. 3D and E). We next
visualized autophagosome-like structures in HFFs treated with
the Xpo-t -specific siRNA. We observed a comparable increase
(∼2-fold) of autophagosomes upon downregulation of Xpo-t
in cells grown in rich or nutrient-depleted medium. These data
demonstrate that the reduced expression of Xpo-t mimics the
effect of nutrient limitation on the induction of autophagy.
Amino acid starvation is signaled through tRNA accumu-
lation in the nucleus. We reported here that, similarly to yeast
cells, human fibroblasts also accumulate tRNAs in the nucleus
when tRNA transport is disrupted as a result of reduced expres-
sion of the tRNA-specific karyopherin Xpo-t. Furthermore, our
study revealed changes in activity of the mTORC1 nutrient-
responsive signaling pathways in cells accumulating tRNAs in
the nucleus. A possible role for tRNAs in triggering a nutrient
starvation response through mTORC1 has been suggested pre-
viously.34-36
Whereas these studies have assessed the effect of
uncharged/mischarged tRNAs accumulating in the cell after
blocking aminoacylation of tRNA, we examined changes in
nutrient-related responses and autophagy upon accumulation of
tRNAs in the nucleus. In agreement with some of these studies,
our work supports a hypothesis that the subcellular distribution
of tRNAs, in addition to their aminoacylation state, may be an
important factor for regulation of nutrient-responsive signaling
pathways in human fibroblasts. Thus, it is possible that the sub-
cellular localization of tRNAs may signal the amino acid avail-
ability through signaling to mTOR pathway. Thus, beside their
essential role in protein synthesis to support cell growth and pro-
liferation, tRNAs also seem to play important roles in improv-
ing cellular homeostasis by modulating signaling pathways and
processes that functions to protects cell from nutrient limitations.
It is interesting to note that tRNA (cytosolic and mitochondrial)
also recently has been shown to be involved in preventing apop-
tosis by binding cytochrome c. Thus, tRNA may be involved in
both apoptosis and autophagy, perhaps depending on their cel-
lular localization.42
The role of tRNA in sensing stress and starvation. The use
of tRNAs as a signal is ancient and variants thereof are found
in all kingdoms. In eukaryotes the important roles of tRNAs
in cell proliferation and stress response have been well demon-
strated. During amino acid starvation uncharged tRNAs directly
bind and activate the eIF2 kinase GCN2,21,22
which in turn ini-
tiates general amino acid control pathway, wherein global pro-
tein synthesis is reduced, allowing cells to adapt to the changing
reduced 2-fold in extracts from cells knocked down for Xpo-t,
but not in those treated with siControl or siKar RNAs (Fig. 3B).
As expected, all samples treated with CHL accumulated p62/
SQSTM due to the blockage of lysosomal degradation of the
auophagy cargo (Fig. 3B). During starvation, the level of p62/
SQSTM in Xpo-t knockdown cells was also reduced as com-
pared to control knocked down cells. These data strongly sup-
port the hypothesis that nuclear accumulation of tRNAs caused
by the reduced level of Xpo-t upregulates autophagy.
Nuclear accumulation of tRNAs caused by Xpo-t deple-
tion alters activity of the nutrient-responsive mTOR path-
way. When nutrients are abundant, the serine/threonine kinase
mTOR is found in its active, phosphorylated at Ser2448, form
(mTORSer2448-P
). As a part of the larger complex, mTORC1, the
active mTOR kinase modulates cell size and growth through
tight control of protein synthesis in response to nutrient availabil-
ity. The mTORC1 pathway stimulates nutrient uptake and pro-
motes ribosome biogenesis and protein synthesis; among mTOR
kinase targets are S6K protein kinase, which phosphorylates
ribosomal protein S6, and the translational inhibitor 4E-BP1
that acts through the initiation factor eIF4E.13
Under amino acid
replete conditions, mTORC1 also inhibits autophagy;32
it has
also been shown that TOR in Drosophila melanogaster directly
phosphorylates Atg1, thereby reducing its activity and subse-
quent autophagy induction.33
During amino acid starvation,
phosphorylation of Ser2448 decreases. This results in reduced
mTOR kinase activity, in turn leading to a decrease in phospho-
(Thr389)-S6K and phospho- (Ser235/236) S6, and subsequent
downregulation of the translational machinery. Conversely, Atg1
undergoes dephosphorylation, becomes activated, and induces
the autophagy cascade. Besides the interaction of the RAG pro-
teins with mTORC1, little is known about how mTORC1 senses
the intracellular levels of amino acids.12,13,32,33
Since amino acid starvation leads to nuclear accumulation of
tRNA, we intended to investigate whether subcellular localiza-
tion of tRNAs may play a role in signaling amino acid avail-
ability. We measured the levels of active mTORSer2448-P
in protein
extracts from fibroblasts knocked down for Xpo-t or the control
karyopherin siKar, as well as cells treated with non-targeting
control siRNA, by immunoblotting (Fig. 3C). Whereas no sig-
nificant changes in mTORSer2448-P
were detected in cells trans-
fected with control or Kar siRNA, a significant reduction was
observed in Xpo-t knocked down cells (Fig. 3C). This strongly
indicates that tRNA accumulation caused by a decrease in the
Xpo-t levels leads to downregulation of the mTORC1 pathway.
To evaluate this indication, we examined the phosphorylation
states of the downstream components of the mTORC1 path-
way S6 (S6Ser235/236-P
) and 4E-BP(4E-BPThr37/46-P
). We observed
a comparable reduction in the levels of phosphorylated forms of
S6 and 4E-BP upon the Xpo-t knock down, whereas the levels
of the unmodified proteins remained the same (Fig. 3C). The
reduced phosphorylation of mTOR, S6 and 4E-BP observed in
cells specifically knocked down for Xpo-t strongly suggests that
the activity of the mTORC1 is downregulated at low levels of
Xpo-t, a condition which leads to tRNA accumulation in the
nucleus.

environment by altering their transcription program. Recent
global analysis of yeast tRNA charging status demonstrated
that the charging pattern changes in response to both nutrient
starvation and osmotic stress.37
Even in the case of amino acid
starvation, the response appears to be global as starvation for a
given amino acid favors deacylation of non-cognate tRNAs. In
HeLa cells, immune response and oxidative stress lead to a ten-
fold increase in misacylation of many tRNA species with Met,
followed by incorporation of Met into growing peptide chains;38
interestingly, misacylation was exclusively cytosolic, highlighting
the importance of subcellular tRNA localization. tRNA modi-
fications, such as thiolation by a ubiquitin-like Urm1p protein,
may be critical to regulate cellular responses to nutrient starvation
and oxidative stress conditions.39
Furthermore, tRNAs can also
be endonucleolytically cleaved by cytoplasmic nucleases (such as
yeast Rny1) during stress, inducing a variety of responses that
may range from GCN2 activation to general translational repres-
sion to induction of siRNA and miRNA pathways.40
In this work, we report that tRNA retention in the nucleus trig-
gers a starvation-like response even under nutrient-replete condi-
tions. This effect could be mediated by nucleus-specific changes
in the retained tRNAs. Modification and cleavage enzymes are
expected to be specifically localized, thus the pattern of tRNA
modifications and the regulatory signals induced by these modi-
fications would also be compartmentalized.
Experimental Procedures
Cells and growth media. Primary human foreskin fibroblast
cells (HFF) were grown in DMEM (Dulbecco’s Modified Eagle’s
Medium, Sigma-Aldrich) supplemented with 10% heat-inacti-
vated fetal bovine serum (FBS, Invitrogen) and 5% Penicillin and
Streptomycin (Sigma-Aldrich). Cells were grown at 37°C with
5% CO2
until 75 to 90% confluent. HFF cells were starved (6 h)
by incubation in Earle’s Balanced Salt Solution (Sigma-Aldrich).
Chloroquine 40 µM was added 3 h prior to harvesting the fibro-
blasts, cells were washed twice with PBS (Phosphate Buffered
Saline, pH 7.4) and detached by incubating with Trypsin (0.25%
Trypsin-EDTA solution, Sigma Aldrich) for 1–3 minutes at 37°C.
Cells were collected by centrifugation at 1,250 rpm for 8 minutes.
siRNA-mediated knock-down. Cells were harvested and
concentrated to 2 x 105
/ml prior to nucleofection, which were
done according to manufacturer’s nucleofection protocol (Amaxa
Nucleofector, Lonza) using 1.5 µg siRNA duplex specific to
TNPO3 (sense 5'-CUG AAU UAC UGC CGU AUU U and
antisense 5'-AAA UAC GGC AGU AAU UCA G), Xpo-t (oligo
1: sense 5'-GAU AGU UAG UUG GAG UAA A and antisense
5'-UUU ACU CCA ACU AAC UAU C and oligo 2: sense 5'-CCU
ACU UCA UGA UCA UGA A and antisense 5'-UUC AUG AUC
AUG AAG UAG G). The latter was used to repeat experimemts
to verify that results obtained with Xpo-t oligo 1 are specific for
knock-down of the karyoperin [Data not shown], non-targeting
scrambled siRNA (5'-GAU CAU ACG UGC GAU CAG ATT
and Antisense 5'-UCU GAU CGC ACG UAU GAU CTT) or
siRNA towards luciferase (Sense 5'-AAA CAU GCA GAA AAU
GCU G and Antisense 5'-CAG CAU UUU CUG CAU GUU U)
as controls (Sigma Aldrich). 2 x 106
cells were used per nucleo-
fection. Cells were incubated 24 h (experiments that include
24 hours starvation) or 48 hours before they were used in the
described experiments.
Isolation of nuclear and cytoplasmic RNA. RNA was iso-
lated using a Cytoplasmic and Nuclear RNA Purification Kit
(Norgen, Canada) using the manufacturer’s protocol. The RNA
was used for quantitative RT-PCR as described below.
Quantitative RT-PCR. Cells were harvested in Trizol Reagent
(Invitrogen), snap frozen to inhibit activity of endogenous RNases
where after RNA was extracted according to the manufacturer’s
protocol (Invitrogen). One µg of total RNA was used to reverse
transcribe mRNA into cDNA using random hexamer primers
and M-MLV Reverse Transcriptase (Invitrogen). qPCR (SYBR®
Green I dye detection, Applied Biosystems, WA) was performed
in triplicate on a sequence detection system (Prism 7300; Applied
Biosystems) using SABiosciences primers to human XPOT
(NM_007235.3 position 2551–2571); GADPH was used as an
internal control (sense, 5'-CCC CTT CAT TGA CCT CAA
CTA CAT-3' and antisense, 5'-CGC TCC TGG AAG ATG
GTG A-3'). Oligos towards tRNALys
were sense 5'-AGC TCA
GTC GGT AGA GCA TGA-3' and antisense 5'-AAC CCA CG
ACC CTG AGA TTA A. XPOT and tRNALys
expressions were
normalized to GADPH.
Immunoblotting. Cell extracts were prepared in 1X RIPA
Buffer (Thermo Scientific) supplemented with Halt Phosphatase
Inhibitor Cocktail and Protease Inhibitor Cocktail EDTA-free
(Thermo Scientific). Proteins from cell extracts were resolved
by SDS PAGE (6, 10 and 13%) and transferred onto 0.45 µm
Pure Nitrocellulose membrane (Bio-Rad). The protein blots
were incubated with indicated antibodies in PBS-T buffer (8 g
NaCl, 0.2 g KCL, 1.44 g Sodium Phosphate, 0.24 g Potassium
Phosphate per 1 L and 0.1% Tween 20 (Sigma-Aldrich) and 3%
BSA (Albumin from bovine serum, Sigma-Aldrich). Monoclonal
anti-Actin, anti-LC3B and anti-Xpo-t antibodies were purchased
from Sigma-Aldrich, anti-p62/SQSTM from MBL and anti-
mTOR, phospho-mTOR (Ser2448), phospho-S6 (Ser235/236),
AKT1, phosphor-AKT1 (Ser473), and 4E-BPThr37/46
were pur-
chased from Cell Signaling Technology. Goat anti-rabbit and
goat anti-mouse Infrared IRDye®
-labeled secondary antibod-
ies (LI-COR) were used. Proteins recognized by the antibodies
were detected using the Detection LI-COR/Odyssey Infrared
Imaging System.
Fluorescent in-situ hybridization. HFF cells transfected
with non-targeting siRNA or siRNA towards Xpo-t were grown
on cover slips in non-starvation media or starvation media.
Cells were then fixed and processed as previously described.41
Dig-labeled hybridization probes used were complementary to
human tRNACUU
Lys
(CCA ACG TGG GGC TCG AAC CCA
CGA CCC TGA GAT TAA GAG TCT CAT GCT CTA CCG
ACT) and as negative control to D. discoidium tRNAGlu
(CCA
GTG TTA GAG ACT AGA GTG TAC CGA CTA CAC CAA
TGA) and were previously described.28
The oligonucleotides
were from Sigma-Aldrich and FITC-conjugated anti-DIG anti-
bodies were from Roche. Fluorescent images were observed by
using an Olympus FV1000-Filter Confocal Microscope (Japan)

and FITC intensity in the cytoplasm and nucleus quantified from
3 images per condition using NIS-Elements BR software.
Transmission electron microscopy. Monolayer cells were
grown in chamber slides in non-starvation or starvation media.
Prior to processing the cells were fixed for 30 min in 2.5% glu-
taraldehyde in 0.1 M phosphate buffer pH 7.4. Further process-
ing was performed by OSU Campus Microscopy and Imaging
Facility. Images were obtained with a FEI Technai G2
Spirit
Transmission Electron Microscope.
Acknowledgements
We would like to thank Irina Artsimovitch for her remarkable
assistance and valuable comments and suggestions in writing
this manuscript. We greatly appreciate Michael Ibba and Jerneja
Tomsic for suggestions and critically reading of the manuscript.
Daniel Klionsky is acknowledged for providing the plasmid
expressing GFP-Atg8 and Anita Hopper for yeast strains, Gustavo
Leone for the HFF cell line and Rebecca Hurto for advice con-
cerning the fluorescent in situ hybridization experiment. OSU
Campus Microscopy and Imaging Facility is acknowledged for
sample preparation and assistance with transmission electron
microscopy, confocal and fluorescent microscopy and Randy
J. Giedt and Rita Alevriadou for quantification of fluorescence
intensities. The work is support by seed grants from OSU
Comprehensive Cancer Center, an OSU Critical Difference for
Women Award and a Beginning Grant-In-Aid from American
Heart Association #09BGIA2230347.
Note
Supplementry materials can be found at:
www.landesbioscience.com/supplement/HuynhCC9-15-sup.pdf
References
1. Gustafsson AB, Gottlieb RA. Autophagy in ischemic
heart disease. Circ Res 2009; 104:150-8.
2. Nishida K, Kyoi S, Yamaguchi O, Sadoshima J, Otsu
K. The role of autophagy in the heart. Cell Death
Differ 2009; 16:31-8.
3. Karantza-Wadsworth V, Patel S, Kravchuk O, Chen G,
Mathew R, Jin S, et al. Autophagy mitigates metabolic
stress and genome damage in mammary tumorigenesis.
Genes Dev 2007; 21:1621-35.
4. Levine B, Deretic V. Unveiling the roles of autophagy
in innate and adaptive immunity. Nat Rev Immunol
2007; 7:767-77.
5. Mizushima N, Levine B, Cuervo AM, Klionsky DJ.
Autophagy fights disease through cellular self-digestion.
Nature 2008; 451:1069-75.
6. Shintani T, Klionsky DJ. Autophagy in health and dis-
ease: a double-edged sword. Science 2004; 306:990-5.
7. He C, Klionsky DJ. Regulation mechanisms and sig-
naling pathways of autophagy. Annu Rev Genet 2009;
43:67-93.
8. Amaravadi RK, Thompson CB. The roles of therapy-
induced autophagy and necrosis in cancer treatment.
Clin Cancer Res 2007; 13:7271-9.
9. White E. Autophagic cell death unraveled:
Pharmacological inhibition of apoptosis and autophagy
enables necrosis. Autophagy 2008; 4:399-401.
10. Kourtis N, Tavernarakis N. Autophagy and cell death
in model organisms. Cell Death Differ 2009; 16:21-30.
11. Scarlatti F, Granata R, Meijer AJ, Codogno P. Does
autophagy have a license to kill mammalian cells? Cell
Death Differ 2009; 16:12-20.
12. Sancak Y, Peterson TR, Shaul YD, Lindquist RA,
Thoreen CC, Bar-Peled L, et al. The Rag GTPases bind
raptor and mediate amino acid signaling to mTORC1.
Science 2008; 320:1496-501.
13. Meijer AJ, Codogno P. Nutrient sensing: TOR’s
Ragtime. Nat Cell Biol 2008; 10:881-3.
14. Sancak Y, Sabatini DM. Rag proteins regulate amino-
acid-induced mTORC1 signalling. Biochem Soc Trans
2009; 37:289-90.
15. Cook A, Bono F, Jinek M, Conti E. Structural biology
of nucleocytoplasmic transport. Annu Rev Biochem
2007; 76:647-71.
16. Cook AG, Fukuhara N, Jinek M, Conti E. Structures
of the tRNA export factor in the nuclear and cytosolic
states. Nature 2009; 461:60-5.
17. Hellmuth K, Lau DM, Bischoff FR, Kunzler M, Hurt
E, Simos G. Yeast Los1p has properties of an exportin-
like nucleocytoplasmic transport factor for tRNA. Mol
Cell Biol 1998; 18:6374-86.
18. Kohler A, Hurt E. Exporting RNA from the nucleus to
the cytoplasm. Nat Rev Mol Cell Biol 2007; 8:761-73.
19. Grosshans H, Hurt E, Simos G. An aminoacylation-
dependent nuclear tRNA export pathway in yeast.
Genes Dev 2000; 14:830-40.
20. Grosshans H, Simos G, Hurt E. Review: transport of
tRNA out of the nucleus-direct channeling to the ribo-
some? J Struct Biol 2000; 129:288-94.
21. Wek SA, Zhu S, Wek RC. The histidyl-tRNA synthe-
tase-related sequence in the eIF-2alpha protein kinase
GCN2 interacts with tRNA and is required for activa-
tion in response to starvation for different amino acids.
Mol Cell Biol 1995; 15:4497-506.
22. Hinnebusch AG. Translational regulation of GCN4
and the general amino acid control of yeast. Annu Rev
Microbiol 2005; 59:407-50.
23. Dever TE, Feng L, Wek RC, Cigan AM, Donahue TF,
Hinnebusch AG. Phosphorylation of initiation factor 2
alpha by protein kinase GCN2 mediates gene-specific
translational control of GCN4 in yeast. Cell 1992;
68:585-96.
24. Vattem KM, Wek RC. Reinitiation involving upstream
ORFs regulates ATF4 mRNA translation in mammali-
an cells. Proc Natl Acad Sci USA 2004; 101:11269-74.
25. Hinnebusch AG. Translational regulation of yeast
GCN4. A window on factors that control initiator-
trna binding to the ribosome. Journal of Biological
Chemistry 1997; 272:21661-4.
26. Takano A, Endo T, Yoshihisa T. tRNA actively shuttles
between the nucleus and cytosol in yeast. Science 2005;
309:140-2.
27. Shaheen HH, Hopper AK. Retrograde movement
of tRNAs from the cytoplasm to the nucleus in
Saccharomyces cerevisiae. Proc Natl Acad Sci USA 2005;
102:11290-5.
28. Shaheen HH, Horetsky RL, Kimball SR, Murthi A,
Jefferson LS, Hopper AK. Retrograde nuclear accumu-
lation of cytoplasmic tRNA in rat hepatoma cells in
response to amino acid deprivation. Proc Natl Acad Sci
USA 2007; 104:8845-50.
29. Pankiv S, Clausen TH, Lamark T, Brech A, Bruun
JA, Outzen H, et al. p62/SQSTM1 binds directly to
Atg8/LC3 to facilitate degradation of ubiquitinated
protein aggregates by autophagy. Journal of Biological
Chemistry 2007; 282:24131-45.
30. Komatsu M, Waguri S, Koike M, Sou YS, Ueno
T, Hara T, et al. Homeostatic levels of p62 control
cytoplasmic inclusion body formation in autophagy-
deficient mice. Cell 2007; 131:1149-63.
31. Bjorkoy G, Lamark T, Pankiv S, Overvatn A, Brech
A, Johansen T. Monitoring autophagic degradation of
p62/SQSTM1. Methods Enzymol 2009; 452:181-97.
32. Diaz-Troya S, Perez-Perez ME, Florencio FJ, Crespo JL.
The role of TOR in autophagy regulation from yeast to
plants and mammals. Autophagy 2008; 4:851-65.
33. Chang YY, Juhasz G, Goraksha-Hicks P, Arsham AM,
Mallin DR, Muller LK, et al. Nutrient-dependent regu-
lation of autophagy through the target of rapamycin
pathway. Biochem Soc Trans 2009; 37:232-6.
34. Iiboshi Y, Papst PJ, Kawasome H, Hosoi H, Abraham
RT, Houghton PJ, et al. Amino acid-dependent control
of p70(s6k). Involvement of tRNA aminoacylation in
the regulation. Journal of Biological Chemistry 1999;
274:1092-9.
35. Wang X, Fonseca BD, Tang H, Liu R, Elia A, Clemens
MJ, et al. Re-evaluating the roles of proposed modula-
tors of mammalian target of rapamycin complex 1
(mTORC1) signaling. Journal of Biological Chemistry
2008; 283:30482-92.
36. Arsham AM, Neufeld TP. A genetic screen in
Drosophila reveals novel cytoprotective functions of
the autophagy-lysosome pathway. PLoS One 2009;
4:6068.
37. Zaborske JM, Narasimhan J, Jiang L, Wek SA, Dittmar
KA, Freimoser F, et al. Genome-wide analysis of tRNA
charging and activation of the eIF2 kinase Gcn2p. J
Biol Chem 2009; 284:25254-67.
38. Netzer N, Goodenbour JM, David A, Dittmar KA,
Jones RB, Schneider JR, et al. Innate immune and
chemically triggered oxidative stress modifies transla-
tional fidelity. Nature 2009; 462:522-6.
39. Leidel S, Pedrioli PG, Bucher T, Brost R, Costanzo M,
Schmidt A, et al. Ubiquitin-related modifier Urm1 acts
as a sulphur carrier in thiolation of eukaryotic transfer
RNA. Nature 2009; 458:228-32.
40. Thompson DM, Parker R. The RNase Rny1p cleaves
tRNAs and promotes cell death during oxidative stress
in Saccharomyces cerevisiae. J Cell Biol 2009; 185:43-50.
41. Matera AG, Ward DC. Nucleoplasmic organization of
small nuclear ribonucleoproteins in cultured human
cells. J Cell Biol 1993; 121:715-27.
42. Mei Y, Yong J, Liu H, Shi Y, Meinkoth J, Dreyfuss G,
et al. tRNA binds to cytochrome c and inhibits caspase
activation. Mol Cell 2010; 37:668-78.

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