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Proc Natl Acad Sci U S A. May 22, 2007; 104(21): 8845–8850.
Published online May 14, 2007. doi:  10.1073/pnas.0700765104
PMCID: PMC1868590
Cell Biology

Retrograde nuclear accumulation of cytoplasmic tRNA in rat hepatoma cells in response to amino acid deprivation


Until recently, transport of tRNA was presumed to be unidirectional, from the nucleus to the cytoplasm. Our published findings, however, revealed that cytoplasmic tRNAs move retrograde to the nucleus in Saccharomyces cerevisiae and that nuclear accumulation of cytoplasmic tRNAs occurs when cells are nutrient deprived. The findings led us to examine whether retrograde nuclear accumulation of cytoplasmic tRNAs occurs in higher eukaryotes. Using RNA FISH and Northern and Western analyses we show that tRNAs accumulate in nuclei of a hepatoma cell line in response to amino acid deprivation. To discern whether tRNA nuclear accumulation results from nuclear import of cytoplasmic tRNAs, transcription of new RNAs was inhibited, and the location of “old” tRNAs in response to nutrient stress was determined. Even in the absence of new RNA synthesis, there were significant tRNA nuclear pools after amino acid depletion, providing strong evidence that retrograde traffic is responsible for the tRNA nuclear pools. Further analyses showed that retrograde tRNA nuclear accumulation in hepatoma cells is a reversible and energy-dependent process. The data provide evidence for retrograde tRNA nuclear accumulation in intact mammalian cells and support the hypothesis that nuclear accumulation of cytoplasmic tRNA and tRNA re-export to the cytoplasm may constitute a universal mechanism for posttranscriptional regulation of global gene expression in response to nutrient availability.

Keywords: nucleus, nutrient deprivation, retrograde traffic

In eukaryotes the nuclear envelope separates mRNA transcription from cytoplasmic translation. The nuclear and cytoplasmic compartments interface at the nuclear pores, which regulate transport of molecules in and out of the nucleus. The majority of nuclear/cytoplasmic exchange occurs via a process that requires the small GTPase Ran and members of the β-importin protein family.

With the exception of small nuclear RNA, the transport of RNA transcripts across the nuclear envelope was presumed to be unidirectional, from the nucleus to the cytoplasm (for review see ref. 1). This dogma has now been challenged. Yoshihisa and coworkers (2) showed that tRNA splicing in yeast occurs in the cytoplasm. Yet spliced tRNA can accumulate in the nucleus. Accumulation of spliced tRNA in the nucleus prompted examination of whether cytoplasmic tRNA is imported into the nucleus. Our work (3) and the work of Takano et al. (4) showed that tRNA moves retrograde from the cytoplasm to the nucleus in Saccharomyces cerevisiae.

Retrograde tRNA nuclear import might serve to proofread tRNAs after splicing to separate improperly spliced tRNAs from the translation machinery. If so, then the retrograde process could be restricted to organisms with cytoplasmic pre-tRNA splicing. According to this scenario, vertebrate cells that splice pre-tRNA in the nucleus (57) may not import cytoplasmic tRNAs into their nuclei. However, because some tRNA modifications occur in the cytoplasm (for review see ref. 8) and the long-lived tRNAs could be damaged in the cytoplasm, tRNA proofreading might be required for both organisms that splice pre-tRNAs in the cytoplasm and organisms that splice pre-tRNAs in the nucleus. On the other hand, because we showed that nuclear accumulation of cytoplasmic tRNA occurs when yeast cells are nutrient deprived (3), the retrograde process could serve as a conserved process to down-regulate translation in response to nutrient availability. According to this scenario, vertebrate cells that are well known to respond to nutrient deprivation via inhibition of protein synthesis (for review see ref. 9) may also have the capability to import cytoplasmic tRNA into nuclei. The possibilities of the retrograde tRNA process functioning in tRNA proofreading and/or regulation in response to nutrient availability led us to examine whether the tRNA retrograde process is conserved in vertebrate cells.

We sought to examine whether cytoplasmic tRNA is imported into the nuclei of rat hepatoma cells in culture. Here, we present evidence that cytoplasmic tRNA accumulates in nuclei, in an energy-dependent manner, in response to amino acid starvation in rat hepatoma cells.


tRNA Localization in Rat Hepatoma Cells by RNA FISH.

We previously used a variation of FISH to localize tRNAs in the budding yeast, S. cerevisiae (10). We investigated the possibility of applying similar technology to study tRNA cellular dynamics in rat hepatoma (H4IIE) cells in culture. Even though FISH for H4IIE cells had not been previously reported to our knowledge, we chose to conduct these studies with H4IIE cells because there is a long history of the response of rat liver cells to nutritional conditions using in situ perfusion, whole liver tissue, and these hepatoma cells in culture (1113). tRNA location was assayed by using digoxiginin (DIG)-labeled DNA probes that were designed to hybridize with mature rat tRNALys (HUSS122), the intron of rat tRNALeu (HUSS121), or as a negative control, to Dictyostelium discoidium tRNAGlu [FISH02 (3)]. After hybridization with the DIG-labeled probes, cells were stained with FITC-conjugated anti-DIG antibodies, followed by DAPI staining to visualize nuclei.

No signal was detected in cells treated with hybridization mix lacking any oligonucleotide probe or cells hybridized with the nonspecific FISH02 probe (data not shown). Hybridization with the tRNA intron-specific tRNALeu probe (HUSS121) resulted in staining in a subregion of the nucleus (Fig. 1A a and a′). In contrast to the subnuclear location of intron-containing tRNAs, cells incubated with a probe specific to mature tRNA (HUSS122) exhibited both nucleoplasmic and cytoplasmic staining (Fig. 1A b and b′). The staining pattern for mature tRNA is very similar to the rather even distribution of signal obtained for budding yeast when mature tRNAs are located by FISH (3, 10). The lack of signal in the absence of probe or when using a nonspecific probe and the different subcellular distributions of intron-containing pre-tRNA vs. mature tRNA document that the FISH procedure used here for rat hepatoma cells is specific and can be used to study tRNA dynamics in vertebrate cells.

Fig. 1.
FISH analysis of the distribution of tRNAs in fed and nutrient-deprived rat hepatoma cells. (A) Detection of rat hepatoma tRNAs by FISH. H4IIE rat hepatoma cells (2 × 105) were grown in complete medium for 72 h and shifted to complete medium for ...

Effect of Amino Acid Deprivation on tRNA Distribution in Rat Hepatoma Cells.

Cytoplasmic tRNAs have been shown to accumulate in the nuclei of S. cerevisiae in response to amino acid deprivation (3). We used FISH to learn whether cytoplasmic tRNAs also accumulate in nuclei in cultured rat hepatoma cells. Cells grown in complete medium were transferred to medium lacking all amino acids and incubated for 2.5 h at 37°C. To verify that this regimen effectively induced nutrient deprivation we monitored 4E-BP1 phosphorylation. Previous studies have shown 4E-BP1 phosphorylation to be reduced in a variety of cell types in response to amino acid deprivation and increased upon their readdition to the culture medium (14, 15). Therefore, in the present study, changes in 4E-BP1 phosphorylation were used as an index of the efficacy of amino acid deprivation and readdition. The phosphorylation state of 4E-BP1 can conveniently be measured as changes in mobility during SDS/PAGE whereby phosphorylation leads to a decrease in migration. As shown in Fig. 1B, in cells maintained in complete medium 4E-BP1 was primarily in the β- and γ-forms. However, in cells deprived of amino acids, the distribution of 4E-BP1 changed, such that the majority of the protein was present in the α- and β-forms, indicating that 4E-BP1 phosphorylation was decreased in amino acid-deprived cells compared with control cells.

The location of tRNALys was subsequently monitored by FISH analysis. Shifting the cells to a medium lacking all amino acids resulted in a strong nuclear signal, suggesting that the tRNALys cellular pool became nuclear in response to amino acid deprivation (Fig. 1A c and c′). The nuclear accumulation of tRNALys during amino acid starvation in rat hepatoma cells is reminiscent of the manner tRNAs redistribute in yeast during nutrient deprivation (3).

We also investigated the location of tRNALys in amino acid-starved cells after nutrient replenishment. Rat hepatoma cells were grown for 2 h in medium lacking all amino acids and then were shifted to a complete medium for 30 min. Readdition of amino acids to deprived cells resulted in a return of 4E-BP1 phosphorylation to the control pattern (Fig. 1B). These results demonstrate the effectiveness of amino acid readdition in H4IIE cells. Upon readdition of amino acids mature tRNA signal was homogeneously distributed throughout the cells (Fig. 1A d and d′). The results indicate that the aberrant nuclear tRNA location under nutrient deprivation is reversible upon nutrient provision, as we also have shown for budding yeast (26).

tRNA Nuclear Import in Rat Hepatoma Cells.

We (3) and Takano et al. (4) originally proposed that tRNAs move retrograde from the cytoplasm to the nucleus because in yeast intron removal occurs in the cytoplasm, yet mature tRNAs accumulate in the nucleus in certain mutants unable to aminoacylate tRNAs or under conditions where cells are deprived for amino acids (3, 1618). Using a combination of FISH and heterokaryon analysis we showed that a reporter tRNA can be recruited from the joint cytoplasm of heterokaryons to nuclei that do not encode the reporter tRNA, proving that tRNAs move retrograde from the cytoplasm to the nucleus (3). Takano et al. (4) came to a similar conclusion by using RNA polymerase inhibitors and showing that tRNA nuclear accumulation occurs in the absence of newly transcribed tRNA.

The mature tRNAs that accumulate in nuclei of rat hepatoma cells in response to amino acid deprivation could have been derived from the cytoplasm by an analogous retrograde process. Alternatively, accumulation of mature tRNAs in rat nuclei during amino acid starvation could have resulted from failure to export newly transcribed tRNA. Because the mammalian tRNA splicing endonuclease complex is localized in the nucleus (7), accumulation of spliced tRNAs in mammalian nuclei cannot be used to judge whether tRNA is imported from the cytoplasm to nuclei of rat hepatoma cells. To distinguish whether the nuclear pool of tRNALys in starved rat hepatoma cells originated from the cytoplasm, rather than being a result of a block of the primary tRNA export pathway, we prevented synthesis of new tRNA by using an inhibitor of transcription. We treated cells with actinomycin D (ActD) to block de novo RNA synthesis. Cells were then transferred to either complete media or media lacking all amino acids in the continued presence of ActD. Cells grown in the absence of ActD were assayed as a control. These cultures were used to prepare protein extracts for Western analysis and total RNA for Northern analysis, or they were fixed and subjected to FISH analysis.

We reasoned that levels of intron-containing tRNA species could be used as a measure of newly synthesized tRNA. Transcription generates transient pre-tRNAs that are processed to mature species that have very long half-lives. Thus, exposure of cells to short periods of transcription inhibition should result in the disappearance of pre-tRNAs without affecting the pool of mature tRNAs. To determine whether ActD is sufficient to inhibit new tRNA synthesis in rat hepatoma cells, we used Northern analysis using a 32P labeled probe specific to the intron-containing precursor of tRNACAALeu (Fig. 2Aa). A 32P-labeled probe to tRNACAGLeu, encoded by a gene lacking an intron, served to ensure equal loading (Fig. 2Ab). As anticipated, we detected intron-containing pre-tRNACAALeu in RNA isolated from cells grown in the absence of ActD when cells were grown in rich, amino acid-deprived, or amino acid-replenished conditions (Fig. 2Aa, lanes 1, 2, and 3, respectively; and corresponding tRNACAALeu bands in Fig. 2b, lanes 1′, 2′, and 3′, respectively). In contrast, no pre-tRNACAALeu was detected from RNA isolated from cells under any of the conditions after ActD addition (Fig. 2Aa lanes 4, 5, and 6; and corresponding tRNACAALeu band in Fig. 2Ab, lanes 4′, 5′, and 6′, respectively). The data show that transcription of tRNAs was inhibited after treatment with ActD.

Fig. 2.
Import of cytoplasmic tRNAs into rat hepatoma nuclei. (A) Northern analysis performed on total RNA isolated from rat hepatoma cells by probing for tRNALeu intron (a) or mature tRNALeu (b). H4IIE cells were grown in complete medium for 72 h then shifted ...

To determine whether or not ActD altered amino acid signaling in H4IIE cells 4E-BP1 phosphorylation was assessed in control cells and cells treated with ActD. As shown in Fig. 2B, 4E-BP1 was predominantly present in the β- and γ-forms regardless of whether or not cells in complete media were exposed to ActD. Moreover, ActD had no effect on the amino acid deprivation-induced dephosphorylation of 4E-BP1. Finally, readdition of amino acids to deprived cells restored 4E-BP1 phosphorylation to control values in both the presence and absence of ActD. Overall, the results show that in H4IIE cells ActD does not interfere with amino acid signaling to 4E-BP1.

FISH analysis showed that tRNALys appeared cytoplasmic and excluded from the nuclei when cells were incubated in complete medium in the presence of ActD (Fig. 2C b and b′), consistent with the inhibition of newly synthesized tRNAs residing in the nucleus and in sharp contrast to the homogeneous cellular distribution of tRNALys observed when cells were grown in complete medium in the absence of ActD (Fig. 2C a and a′). The data verify inhibition of tRNA transcription by ActD. Importantly, a significant nuclear pool of tRNALys was detected in the nuclei when cells were incubated in medium lacking all amino acids in the presence of ActD (Fig. 2C c and c′). The observation that tRNALys is significantly nuclear after amino acid starvation, even in absence of de novo tRNA synthesis, argues in favor of the hypothesis that cytoplasmic tRNALys accumulates in nuclei of rat hepatoma cells in response to amino acid deprivation.

The tRNAs accumulating in the nucleus in yeast during nutrient deprivation have been shown to be re-exported back to the cytoplasm upon nutrient replenishment (26). We monitored the dynamics of tRNALys in amino acid-starved H4IIE cells that were treated with ActD, and then subsequently shifted back to complete medium containing ActD for 30 min. Instead of the “void” nuclei that are present in nonstarved cells that were treated with ActD (Fig. 2C b and b′), the cellular distribution of tRNALys appeared homogeneous upon readdition of amino acids to the cells (Fig. 2C d and d′). This finding suggests that most, but not all, of the imported tRNAs were re-exported back to the cytoplasm during the period of amino acid readdition to the media.

Energy Requirement for tRNA Retrograde Nuclear Accumulation in Rat Hepatoma Cells.

The presence of a significant imported pool of tRNALys in the rat hepatoma nucleus after amino acid deprivation indicates that efficient tRNA import machinery may be required for tRNA subcellular dynamics. To discern whether an active, energy-dependent mechanism is required, we used sodium azide, an inhibitor of ATP hydrolysis.

Cells were treated with ActD to inhibit synthesis of new tRNAs for 45 min, followed by a 15-min treatment with sodium azide. The cells were then transferred to either complete medium containing ActD and sodium azide and incubated for an additional 1 h at 37°C or medium lacking all amino acids but containing ActD and sodium azide. The location of tRNALys in rat hepatoma cells under these conditions was compared with untreated cells (Fig. 3A).

Fig. 3.
Import of cytoplasmic tRNA in rat hepatoma nuclei in response to amino acid starvation is inhibited by sodium azide. (A) (a, a′, b, and b′) FISH analysis was performed by probing for tRNALys in cells that were grown in complete medium ...

Previous studies have shown that inhibition of mitochondrial metabolism interferes with amino acid signaling through mammalian target of rapamycin (mTOR) to downstream targets such as 4E-BP1 and the ribosomal protein S6 kinase, S6K1 (19). In agreement with such observations, exposure of cells maintained in complete medium to a combination of ActD and sodium azide resulted in a relative decrease of the γ-phosphorylated form of 4E-BP1 compared with the relative level of γ-4E-BP1 in cells exposed to ActD alone (compare Fig. 2B, lane 4 with Fig. 3B, lane 3). Moreover, in cells deprived of amino acids, treatment with ActD and sodium azide resulted in a similar distribution of the phosphorylated forms of 4E-BP1 as in cells treated with ActD alone (Fig. 3B), suggesting that the combination of ActD and sodium azide does not reverse the effects of amino acid deprivation on signaling.

Untreated cells exhibited a homogeneous tRNALys distribution when grown in complete medium or nuclear accumulation of this tRNA when shifted to medium lacking amino acids (Fig. 3A a and a′ and b and b′, respectively). Treatment with sodium azide alone resulted in a significant nuclear pool of tRNALys whether cells were grown in complete medium or one lacking all amino acids (Fig. 3A c and c′ and d and d′, respectively). Nuclear tRNA accumulation under these conditions is likely caused by inhibition of nuclear export of newly transcribed tRNAs. In contrast, we observed void nuclei in cells that were grown in complete medium containing ActD and then subjected to sodium azide treatment regardless of the presence (Fig. 3A e and e′) or absence (Fig. 3A f and f′) of amino acids in the media. This observation indicates that tRNA nuclear import in response to amino acid deprivation is inhibited in the presence of sodium azide, presumably because nuclear accumulation of cytoplasmic tRNA in rat hepatoma cells requires ATP hydrolysis.


Our previous report of retrograde movement of cytoplasmic tRNAs into the nucleus in yeast (3) raised the question of whether a similar process occurs in higher eukaryotes. Here, we present evidence that nuclear import of tRNA is conserved in rat hepatoma cell culture. Furthermore, imported tRNAs that accumulated in the nucleus in response to nutrient deprivation are re-exported back to the cytoplasm upon nutrient replenishment, as happens in yeast (26). Our findings also support previous observations that energy is required for retrograde tRNA nuclear accumulation (4). Thus, even though the subcellular location of pre-tRNA splicing differs between budding yeast and vertebrate cells, the retrograde tRNA nuclear import process is conserved.

What purpose does retrograde tRNA nuclear import serve for vertebrate cells? One intriguing possibility is that nuclear accumulation of cytoplasmic tRNA serves to separate tRNAs from the protein synthesis machinery under conditions unfavorable for protein synthesis. In both yeast and vertebrate cells, amino acid deprivation results in inhibition of general protein synthesis while stimulating translation of particular proteins responsible for coping with stress conditions. One mechanism to achieve the regulation is via phosphorylation and inhibition of the translation initiation factor eIF2 (for reviews see refs. 9, 20, and 21). Another means to this end in yeast is disassembly of polysomes. In yeast, under conditions of nutrient deprivation (glucose limitation), mRNAs are recruited from polyribosomes to P-bodies (22), thereby separating mRNAs from the protein synthesis machinery. Likewise, retrograde tRNA nuclear import may serve a regulatory role, separating tRNAs from the translation machinery, aiding in down-regulation of translation.

Although tRNA nuclear accumulation serving as a means to regulate posttranscriptional gene expression remains an unproven possibility, recent data indicate that retrograde tRNA nuclear accumulation may occur in vertebrate cells in response to a different stress, viral infection. Zaitseva et al. (23) used reconstituted nuclear import assays with HeLa and 293T cells permeabilized with digitonin to show that the HIV-1 reverse transcriptase complex (RTC) exploits nuclear import of tRNAs in human cells to gain access to the nucleoplasm of infected cells. They also showed that tRNAs with defective 3′ termini were imported into the nucleus, independent of the RTC (23). Retrograde accumulation of tRNAs during viral infections and nutrient deprivation raises the possibility that tRNA nuclear import could be up-regulated during cellular stress.

Zaitseva et al. (23) reported that only tRNAs lacking mature 3′ CCA termini could be imported into nuclei in the permeabilized cell assay. Import of defective tRNAs is also in agreement with the notion that the retrograde pathway may serve a role in tRNA quality control. Accordingly, tRNA nuclear import would ensure that unfit tRNAs are sequestered in the nucleus to prevent interaction with the translation machinery. It is still unclear whether functional tRNAs are also imported into the nucleus. Evidence in yeast suggests that tRNAs are constitutively imported into the nucleus in fed and nutrient deprived cells (H.H.S., A.M., E. M. Phizicky, and A.K.H., unpublished work), which would mean that nuclear re-export of imported tRNAs could be subject to modulation. Thus, under nutrient deprivation nuclear export would be blocked and result in nuclear accumulation of tRNAs. According to this model, defective tRNAs may be retained in the nuclei, whereas functional ones would be re-exported, which may explain why nuclear accumulation of functional tRNAs could not be observed in the HIV-1 report (23). A combination of import and re-export regulation, although hard to distinguish, is also plausible.

What cellular machinery is involved in nuclear import of tRNAs? We show that tRNA retrograde nuclear accumulation in H4IIE cells is inhibited by sodium azide, although it is unknown whether energy is required for signal transduction of nutrient deprivation or the nuclear import process per se. However, others (4, 23) have concluded that tRNA retrograde nuclear import process requires energy. Our work in yeast implicated the Ran-dependent Mtr10 β-importin in tRNA nuclear import in response to nutrient deprivation (3). However, Ran-independent tRNA nuclear import appears to occur under other conditions (4, 23). It will be interesting to learn whether the Ran pathway is involved in tRNA nuclear accumulation in response to nutrient deprivation in rat hepatoma cells or whether it occurs in a Ran-independent fashion.

The conservation of tRNA nuclear import pathway among yeast, rats, and humans emphasizes the biological relevance of this previously unanticipated dynamics. The involvement of tRNA nuclear import in HIV infectivity makes it imperative to determine which cellular factors contribute to the tRNA nuclear import and re-export processes.

Materials and Methods

Cell Culture.

These studies used rat H4IIE cells (13). Coverslips were placed in each well of a six-well plate before addition of H4IIE cells (2 × 105). Cells were maintained for 72 h in DMEM supplemented with 10% heat-inactivated FBS, 100 units/ml benzylpenicillin, and 100 μg/ml streptomycin sulfate. On the day of the study, cells were washed twice with 2 ml Dulbecco's PBS (D-PBS), and then randomly divided into three groups. The first group of cells was incubated for 2.5 h in DMEM supplemented with 10% heat-inactivated FBS, washed twice in ice-cold PBS, and fixed by incubation in 4% paraformaldehyde. The second group of cells was incubated for an equal length of time in DMEM without serum or amino acids and then washed and fixed as above. The third group of cells was incubated for 2 h in DMEM lacking serum and amino acids, at which time the culture medium was removed and medium containing both serum and amino acids was added to the wells. Thirty minutes later, coverslips were removed from the wells and washed with PBS, and the cells were fixed with paraformaldehyde. When present, ActD was added to a final concentration of 5 μg/ml, 120 min before the D-PBS wash. Alternatively, sodium azide was added to some wells at a final concentration of 10 mM, 75 min before the D-PBS wash. In both cases, the drug was also included in the culture medium after washing.


The Pennsylvania State University College of Medicine Macromolecular Core Facility generated all oligonucleotides. Rat tRNA sequences from Todd Lowe (University of California, Santa Cruz) were used to design probes complementary with mature rat tRNACUULys (HUSS122; CCA ACG TGG GGC TCG AAC CCA CGA CCC TGA GAT TAA GAG TCT CAT GCT CTA CCG ACT), mature tRNACAGLeu (HUSS120; AGG AGT GGG ATT CGA ACC CAC GCC TCC AGG GGA GAC TGC GAC CTG AAC GGA GCG CCT), and the intron of rat tRNACAALeu (HUSS121; CAG AAC CCT CAA TGA GAG AAG CCA ACG CTT GAG TCT). FISH02, against D. discoidium tRNAGlu has been described (3).


Cells were fixed in 1 ml of solution A (4% paraformaldehyde/PBS) for 50 min at room temperature and permeabilized in cold acetone for 3 min at −20°C as described (24). FISH was carried out as described (10) with the exception of denaturing target tRNA at 74°C for 5 min after the prehybridization step. Also, cells were prehybridized, hybridized, and washed in 1 ml of the corresponding solutions. Cells were incubated overnight in hybridization solution containing DIG-labeled oligonucleotides: 122 (tRNACUULys), 121 (tRNACAALeu intron), FISH02, against D. discoidium tRNAGlu (3) as a negative control, or no probe at 39°C. Hybridized probes were subsequently stained by using FITC-conjugated anti-DIG.

RNA Isolation and Northern Analysis.

H4IIE cells were incubated as described above, except coverslips were not used. Cell culture medium was removed from the wells by aspiration, and cells were lysed by the addition of 0.75 ml of TRIzol Reagent (Invitrogen, Carlsbad, CA). RNA was then isolated as described in the instructions provided by the manufacturer and resuspended in 20 μl of THE RNA Storage Solution (Ambion, Austin, TX). RNA was separated on 10% polyacrylamide-urea gel, transferred on to a Hybond N+ membrane (Amersham Pharmacia, Piscataway, NJ). ULTRAhyb (Ambion) was used for prehybridization and hybrization, and further Northern analysis was carried out as per manufacturer's instructions with the oligonucleotide HUSS120 and HUSS121.

4E-BP1 Phosphorylation.

Changes in phosphorylation of eIF4E binding protein (4E-BP1) were assessed as altered mobility during SDS/PAGE as described (25). Briefly, cells were harvested in SDS sample buffer, and homogenates were resolved by electrophoresis on 15% polyacrylamide gels. Proteins in the gel were electrophoretically transferred to PVDF membranes (Pall Life Sciences, Exton, PA) and then subjected to Western blot analysis by using an anti-4E-BP1 polyclonal antibody (Bethyl Laboratories, Montgomery, TX). Blots were developed by using a kit (Pierce ECL Western Blotting) from Thermo Fisher Scientific (Waltham, MA) and visualized with a GeneGnome HR imaging system (Syngene, Frederick, MD).


Fluorescence images for FISH studies were observed by using a Microphot-FX microscope (Nikon, Tokyo, Japan). Images were captured by using a SenSys charge-coupled device camera (Photometrics, Tucson, AZ) with QED software (QED Imaging, Pittsburgh, PA). Images were arranged with Photoshop 5.0 (Adobe Systems, San Jose, CA).


We thank M. Whitney and R. Hurto for valuable scientific interactions. This work was supported by National Institutes of Health Grants GM27930 (to A.K.H.) and DK13499 (to L.S.J.).


actinomycin D.


The authors declare no conflict of interest.

This article is a PNAS Direct Submission.


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