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. 2000 Apr 1;14(7):830-40.

An aminoacylation-dependent nuclear tRNA export pathway in yeast

H Grosshans  1 , E Hurt, G Simos
Affiliations

An aminoacylation-dependent nuclear tRNA export pathway in yeast

H Grosshans et al. Genes Dev. .

Abstract

Yeast Los1p, the homolog of human exportin-t, mediates nuclear export of tRNA. Using fluorescence in situ hybridization, we could show that the export of some intronless tRNA species is not detectably affected by the disruption of LOS1. To find other factors that facilitate tRNA export, we performed a suppressor screen of a synthetically lethal los1 mutant and identified the essential translation elongation factor eEF-1A. Mutations in eEF-1A impaired nuclear export of all tRNAs tested, which included both spliced and intronless species. An even stronger defect in nuclear exit of tRNA was observed under conditions that inhibited tRNA aminoacylation. In all cases, inhibition of tRNA export led to nucleolar accumulation of mature tRNAs. Our data show that tRNA aminoacylation and eEF-1A are required for efficient nuclear tRNA export in yeast and suggest coordination between the protein translation and the nuclear tRNA processing and transport machineries.

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Figures

Figure 1
Figure 1
A nuclear tRNA export assay in yeast on the basis of fluorescent in situ hybridization. (a) Localization of mature tRNALeu(CAA) (mature, top) and its corresponding unspliced precursor (intron, bottom) in the wild-type strain RS453 expressing GFP–Nop1p. DNA was visualized by staining with DAPI. The insets in the bottom panel show a single cell in which the signal for tRNA has been colored red and the signal for GFP–Nop1 green so that colocalization is shown by the yellow color upon merging. Representative cells are labeled by arrows that point to the nucleoplasmic space as judged by DAPI staining; arrowheads point to nucleoli as judged by the GFP–Nop1p signal. (b) Localization of tRNAIle(UAU) and tRNAGlu(UUC) in the thermosensitive rna1-1 strain grown at 23°C or shifted for 4 hr to 37°C as indicated. Representative cells are labeled by arrows that point to the nucleoplasmic space as judged by DAPI staining.
Figure 2
Figure 2
Disruption of LOS1 causes nuclear accumulation of a subset of tRNA species. (a) Localization of tRNAGly(GCC) and tRNAGlu(UUC) in the los1− strain grown at 30°C. Representative cells are labeled by arrows that point to the nucleoplasmic space as judged by DAPI staining. (b) Localization of tRNALeu(CAA) (left) and tRNAIle(UAU) (right) in los1− cells grown at 30°C using the mature probes schematically depicted in c. Representative cells are labeled as in a.(c) Schematic representation of mature and intron-containing tRNALeu(CAA) and tRNAIle(UAU). Sequences that hybridize to the probes are indicated by solid circles. In the case of mature tRNALeu(CAA), solid circles represent the sequence hybridizing to probe I, whereas probes II and III are indicated by gray and black lines, respectively. Arrowheads point to splice sites. (d,e) Detection of tRNALeu(CAA) (d) and tRNAIle(UAU) (e) by Northern blot analysis. Total RNA was extracted from wild-type (wt) or los1− cells grown at 30°C. The probes used to detect the tRNAs are indicated below the panels. (d) Formamide (50%) or (e) no formamide were included in the hybridization buffer. (f) Mature and intron-containing tRNALeu(CAA) and tRNAIle(UAU) are localized in los1− and wild-type (wt) cells. Pictures within each box were taken at identical exposure times, that is, 120 msec for tRNALeu(CAA) and 600 msec for tRNAIle(UAU). Probe I was used to detect mature tRNALeu(CAA).
Figure 3
Figure 3
Accumulation of spliced tRNAs in the nucleolus of los1− cells. (Top) Colocalization of tRNALeu(CAA) with GFP–Nop1p in los1− cells grown at 30°C using the mature probe I. A representative cell is labeled by an arrow that points to the nucleoplasmic space as judged by DAPI staining; the arrowhead points to a nucleolus as judged by the GFP–Nop1p signal. (Bottom) A single cell was color coded to allow a merge of the signals for tRNA (red) and Nop1 (green).
Figure 4
Figure 4
eEF-1A is genetically linked to LOS1. (a) The screening strain los1− arc1− plus pGAL–LOS1 (Y1112) transformed with plasmids carrying the indicated genes or mutant alleles was grown on galactose (left) or glucose (right) -containing plates. All genes are on low-copy-number plasmids except TEF1, which is on a high-copy plasmid. (−) Empty plasmid. (b) The double disrupted los1− arc1− strain supplemented with the plasmids pURA–LOS1 and pARC1–ΔC was transformed with the indicated genes on plasmids. To force loss of the pURA–LOS1 plasmid, transformants were subsequently incubated on an FOA-containing plate, on which only ura− cells can grow. (c) The temperature-sensitive double-disrupted los1− pus1− strain was transformed with plasmids carrying the indicated genes and serial dilutions (1:10 steps) of the transformants were spotted on YPD plates and allowed to grow for 2 days at 23°C for 5 days at 37°C. (d) Serial dilutions (1:10 steps) of the indicated strains were spotted on YPD plates and incubated for 2 days at 23°C. (wt) Wild-type parental RS453 strain.
Figure 5
Figure 5
eEF-1A is required for efficient nuclear tRNA export. (a) Localization of tRNALeu(CAA) (probe I) in single disrupted tef2− cells grown at 30°C (first panel from top); in double disrupted tef1− tef2− cells complemented by a plasmid carrying the tef2-1 allele grown at 23°C (second panel); in cells incubated for 60 min in the absence (−CHX) or presence (+CHX) of 10 μg/ml cycloheximide (third panel); in the thermosensitive mutant sui2-1 grown at 23°C or shifted to 37°C for 4 hr as indicated (bottom). (b) Localization of tRNAGly(GCC) in the los1− or tef2− single disruption mutants or the los1− tef2− double disruption mutant. (c) Colocalization of tRNAGly(GCC) and Nop1p in the los1− tef2− double disruption mutant. Color codes are as in Fig. 1. Representative cells are labeled by arrows that point to the nucleoplasmic space as defined by DAPI staining.
Figure 6
Figure 6
Inhibition of tRNA aminoacylation blocks nuclear tRNA export. (a) Localization of tRNALeu(CAA) (probe I) or tRNAIle(UAU) in the leucine auxotrophic wild-type strain RS453 (top) and tRNALeu(CAA) or tRNAGly(GCC) in the isogenic los1− disruption mutant (bottom) grown in complete synthetic medium (+leucine) or shifted for 3 hr to medium lacking leucine (−leucine). (b) Localization of tRNAIle(UAU) and tRNALeu(CAA) in the wild-type strain RS453 grown for 2 hr in complete synthetic medium in the absence (−) or the presence (+) of 500 μg/ml of the isoleucyl-tRNA synthetase inhibitor BAY10-8888. (c) Colocalization of tRNAIle(UAU) and GFP–Nop1p in RS453 cells grown for 2 hr in the presence of 50 μg/ml BAY10-8888. Color codes are as in Fig. 1. (d) Localization of tRNALeu(CAA) (probe I) in the thermosensitive mutant cca1-1 grown at 23°C or shifted to 37°C for 4 hr. Representative cells are labeled by arrows that point to the nucleoplasmic space as defined by DAPI staining.
Figure 7
Figure 7
Unspliced tRNA precursors do not accumulate upon inhibition of nuclear tRNA export. (a) Equal amounts of total RNA extracted from the indicated strains were analyzed by Northern with probes for mature (probe I) or intron containing tRNALeu(CAA). (wt) Wild type grown at 30°C; (tef2−) tef2− grown at 30°C; (tef2-1) tef1− tef2-1 grown at 23°C; (los1) los1− cells grown in the presence (+) or absence (−) of leucine at 30°C; (cca1-1) cca1-1 cells grown at 23°C or shifted to 37°C for 4 hr. The arrowhead indicates tRNA lacking the three terminal CCA nucleotides due to the inactivation of Cca1p. (b) Total RNA was extracted from wild-type cells incubated in the absence (−) or presence (+) of isoleucyl-tRNA synthetase inhibitor and analyzed by Northern with probes for mature or intron-containing tRNAIle(UAU).
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