Retrograde nuclear accumulation of cytoplasmic tRNA in rat hepatoma cells in response to amino acid deprivation - PubMed (original) (raw)

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

Hussam H Shaheen et al. Proc Natl Acad Sci U S A. 2007.

Abstract

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

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 2.5 h. (a, a′, b, and b′) FISH analysis was performed by probing for the intron of tRNALeu (a and a′) or mature tRNALys (b and b′). (c, c′, d, and d′) Alternatively, cells grown in complete medium were either shifted to a medium lacking all amino acids for 2.5 h followed by FISH analysis probing for tRNALys (c and c′) or shifted to a medium lacking all amino acids for 2 h and then incubated in complete medium for 30 min, “add back (AB)” followed by FISH probing for tRNALys (d and d′). (a–d) FITC staining represents tRNA location. (a′–d′) Nuclei were visualized with DAPI staining of DNA for the respective cells. (Scale bar: 5 μm.) (B) H4IIE cells were deprived of amino acids or deprived of amino acids followed by readdition as described above. Cells were harvested, and homogenates were resolved by SDS/PAGE as described in Materials and Methods. The positions of the α-, β-, and γ-forms of 4E-BP1 are denoted to the right of the blot.

Fig. 2.

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 a complete medium for 2.5 h (a, lane 1; b, lane 1′), medium lacking all amino acids for 2.5 h (a, lane 2; b, lane 2′), or medium lacking all amino acids for 2 h followed by amino acid replenishment for 30 min (a, lane 3; b, lane 3′). Alternatively, Northern analysis was performed as above after treating cells with ActD (5 μg/ml) for 1 h then shifting cells to complete medium containing ActD (5 μg/ml) for 2.5 h (a, lane 4; b, lane 4′), medium lacking all amino acids and containing 5 μg/ml ActD for 2.5 h (a, lane 5; b, lane 5′), or medium lacking all amino acids and containing 5 μg/ml ActD for 2.5 h followed by nutrient replenishment in the presence of 5 μg/ml of ActD for 30 min (a, lane 6; b, lane 6′). (B) H4IIE cells were incubated as described above and 4E-BP1 phosphorylation was assessed by Western blot analysis as described in the legend to Fig. 1_B_. The positions of the α-, β-, and γ-forms of 4E-BP1 are denoted to the right of the blot. (C) (a and a′) FISH was performed by probing for tRNALys in H4IIE cells that were grown in complete medium for 72 h then shifted to complete medium for 2 h. (b–d and b′–d′) Cells grown in complete medium for 71 h were then treated with ActD (5 μg/ml) for 1 h followed by transfer to complete medium for 2.5 h (b and b′), medium lacking all amino acids and containing 5 μg/ml of ActD for 2.5 h (c and c′), or medium lacking all amino acids containing of ActD (5 μg/ml) for 2 h followed by replenishment of amino acids by incubating in complete medium containing 5 μg/ml of ActD for 30 min (AB) (d and d′). (a–d) FITC staining represents tRNA location. (a′–d′) Nuclei were visualized with DAPI staining of DNA for the respective cells. (Scale bar: 5 μm.)

Fig. 3.

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 for 72 h then shifted to complete medium for 2.5 h (a and a′) or a medium lacking all amino acids for 2.5 h (b and b′). (c, c′, d, and d′) FISH analysis was performed on cells that were grown for 72 h on complete medium then treated with 10 mM of sodium azide for 15 min and either shifted to a complete medium containing 10 mM sodium azide for 2.5 h (c and c′) or transferred to a medium lacking all amino acids that contained 10 mM of sodium azide for 2.5 h (d and d′). (e, e′, f, and f′) FISH analysis was also performed by probing for tRNALys in cells that were grown on a complete medium for 71 h then treated for 1 h with 5 μg/ml ActD. After 45 min sodium azide was added to the culture medium to a final concentration of 10 mM. Cells were then shifted to complete medium containing ActD and sodium azide (5 μg/ml, 10 mM, respectively) for 2.5 h (e and e′) or medium lacking all amino acids and containing ActD and sodium azide (5 μg/ml, 10 mM, respectively) (f and f′). (a–f) FITC staining represents tRNA location. (a′–f′) Nuclei were visualized with DAPI staining of DNA for the respective cells. (Scale bar: 5 μm.) (B) H4IIE cells were incubated as described above, and 4E-BP1 phosphorylation was assessed by Western blot analysis as described in the legend to Fig. 1_B_. The positions of the α-, β-, and γ-forms of 4E-BP1 are denoted to the right of the blot.

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