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Comparative Study
. 2005 Aug 9;102(32):11290-5.
doi: 10.1073/pnas.0503836102. Epub 2005 Jul 22.

Retrograde movement of tRNAs from the cytoplasm to the nucleus in Saccharomyces cerevisiae

Hussam H Shaheen  1 , Anita K Hopper
Affiliations
Comparative Study

Retrograde movement of tRNAs from the cytoplasm to the nucleus in Saccharomyces cerevisiae

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

Abstract

In eukaryotes, tRNAs transcribed in the nucleus function in cytoplasmic protein synthesis. The Ran-GTP-binding exportin, Los1p/Xpo-t, and additional pathway(s) mediate tRNA transport to the cytoplasm. Although tRNA movement was thought to be unidirectional, recent reports that yeast precursor tRNA splicing occurs in the cytoplasm, whereas fully spliced tRNAs can reside in the nucleus, require that either the precursor tRNA splicing machinery or mature tRNAs move from the cytoplasm to the nucleus. Our data argue against the first possibility and strongly support the second. Combining heterokaryon analysis with fluorescence in situ hybridization, we show that a foreign tRNA encoded by one nucleus can move from the cytoplasm to a second nucleus that does not encode the tRNA. We also discovered nuclear accumulation of endogenous cytoplasmic tRNAs in haploid yeast cells in response to nutritional deprivation. Nuclear accumulation of cytoplasmic tRNA requires Ran and the Mtr10/Kap111 member of the importin-beta family. Retrograde tRNA nuclear import may provide a novel mechanism to regulate gene expression in eukaryotes.

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Figures

Fig. 1.
Fig. 1.
Location of Sen2-GFP under conditions in which mature tRNAs accumulate in the nucleus. (a and a′) American Type Culture Collection strain 201388 possessing the chromosomally located SEN2-GFP allele was grown at 24°C on rich medium, then suspended in 1 μg/ml DAPI in H2O. (b and b′) American Type Culture Collection 201388 in medium lacking leucine for 2 h at 24°C. (c and c′) American Type Culture Collection 201388 cells (TYS1 SEN2-GFP) shifted to 37°C for 2 h. (d and d′) HHS1 (tys1-1 SEN2-GFP) cells shifted to 37°C for 2 h. (a–d) GFP signal. (a′–d′) DAPI signal for mitochondrial and nuclear DNA. (Bar: 5 μm.)
Fig. 2.
Fig. 2.
Specific expression and detection of D. discoideum tRNAGlu in S. cerevisiae. FISH analysis by using a digoxgenin-labled tRNAGlu-D probe in BY4741 with vector alone (a), BY4741 with pRS416tRNAGlu-D (b), MS739 (kar1-1) with pRS416tRNAGlu-D (c), and MS739B (kar1-1 los1Δ) with pRS416tRNAGlu-D (d). (a′–d′) Location of nuclear DNA in the respective cells as determined by DAPI staining. (Bar: 5 μm.)
Fig. 3.
Fig. 3.
FISH analysis showing tRNAGlu-D moves from the shared cytoplasm to the second nucleus of heterokaryon zygotes. (A) los1Δ cells. (a, c, and e) Cells were fixed immediately after zygote formation (T = 0 h) followed by FISH analysis using the tRNAGlu-D probe. (a) LOS1 KAR1 + tRNAGlu-D × LOS1 kar1-1 heterokaryons. (c) los1Δ KAR1+ tRNAGlu-D × LOS1 kar1-1 heterokaryons. (e) Heterokaryon zygote generated by mating los1Δ KAR1 + tRNAGlu-D and los1Δ kar1-1 strains. (a′, c′, and e′) Same cells stained with DAPI to show locations of nuclei. (b, d, and f) Heterokaryons generated as for a, c, and e, fixed after growth for 2 h at 24°C and 2-h incubation at 37°C (T = 2 h). (b′, d′, and f′) Location of nuclear DNA in respective heterokaryons. Arrows point to the two nuclei of a heterokaryon, both containing tRNAGlu-D. (Bar: 5 μm.) (B) Heterokaryons subjected to amino acid deprivation. (a and a′) LOS1 KAR1 + tRNAGlu-D × LOS1 kar1-1 heterokaryons incubated in rich medium at 24°C for 90 min. (b and b′) Heterokaryons of the same genotype as incubated in medium lacking amino acids at 24°C for 90 min. Filled arrows, tRNAGlu-D in two nuclei of a heterokaryon; open arrow, an unmated haploid accumulating tRNAGlu-D under amino acid deprivation. (Bar: 5 μm.)
Fig. 4.
Fig. 4.
Role of the Ran cycle in tRNA nuclear import. (A) FISH analysis to evaluate the dependence of tRNA nuclear import upon the Ran cycle. (a and b) RNA1 KAR1 cells (EE1b-35) were mated in rich medium at 24°C with RNA1 kar1-1 + tRNAGlu-D cells (MS739-35) to generate heterokaryons with two RNA1 nuclei, only one of which encodes tRNAGlu-D. (c and d) rna1-1 KAR1 cells (EE1b-6) were mated with rna1-1 kar1-1 + tRNAGlu-D cells (MS739-6) to generate heterokaryons with two rna1-1 nuclei, only one of which encodes tRNAGlu-D. After 25 min at 37°C, the culture was split, with half incubated 90 min in medium with amino acids (a and c) and half incubated in medium lacking amino acids (b and d), followed by FISH analyses. Filled arrows point to nuclei that accumulate tRNAGlu-D; open arrows point to nuclei with little detectable tRNAGlu-D. (Bar: 5 μm.) (B) Graphic display of tRNA nuclear accumulation. For each mating and condition, ≈50 heterokaryons were assessed and scored as follows: 0, none of the nuclei possessed tRNAGlu-D; 1, one or more of the nuclei in the heterokaryon had no or little tRNAGlu-D signal; 2 or >2, all of the nuclei possessed tRNAGlu-D.
Fig. 5.
Fig. 5.
Analysis of the role of importin-β family members in tRNA nuclear accumulation upon amino acid deprivation. The indicated cell cultures were grown in rich medium, and the cultures were split, with half provided with amino acids and half in medium lacking amino acids. After 2 h, the cultures were processed for FISH. (a–d and a′–d′) WT cells, treated as indicated. (e–h and e′–h′) los1Δ. (i–l and i′–l′) pdr6Δ. (m–p and m′–p′) mtr10Δ. (Bar: 5 μm.)
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Comment in

  • Have tRNA, will travel.
    Phizicky EM. Phizicky EM. Proc Natl Acad Sci U S A. 2005 Aug 9;102(32):11127-8. doi: 10.1073/pnas.0504843102. Epub 2005 Aug 1. Proc Natl Acad Sci U S A. 2005. PMID: 16061803 Free PMC article. Review. No abstract available.

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