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. 2002 Apr 16;99(8):5412-7.
doi: 10.1073/pnas.082682699.

A Los1p-independent pathway for nuclear export of intronless tRNAs in Saccharomycescerevisiae

Wenqin Feng  1 , Anita K Hopper
Affiliations

A Los1p-independent pathway for nuclear export of intronless tRNAs in Saccharomycescerevisiae

Wenqin Feng et al. Proc Natl Acad Sci U S A. .

Abstract

Los1p, the Saccharomyces cerevisiae exportin-t homologue, binds tRNA and functions in pre-tRNA splicing and export of mature tRNA from the nucleus to the cytosol. Because LOS1 is unessential in yeast, other pathways for tRNA nuclear export must exist. We report that Cca1p, which adds nucleotides C, C, and A to the 3' end of tRNAs, is a multicopy suppressor of the defect in tRNA nuclear export caused by los1 null mutations. Mes1p, methionyl-tRNA synthetase, also suppresses the defect in nuclear export of tRNA(Met) in los1 cells. Thus, Cca1p and Mes1p seem to function in a Los1p-independent tRNA nuclear export pathway. Heterokaryon analysis indicates that Cca1p is a nucleus/cytosol-shuttling protein, providing the potential for Cca1p to function as an exporter or an adapter in this tRNA nuclear export pathway. In yeast, most mutations that affect tRNA nuclear export also cause defects in pre-tRNA splicing leading to tight coupling of the splicing and export processes. In contrast, we show that overexpressed Cca1p corrects the nuclear export, but not the pre-tRNA-splicing defects of los1Kan(r) cells, thereby uncoupling pre-tRNA splicing and tRNA nuclear export.

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Figures

Figure 1
Figure 1
Subcellular distribution of tRNAMet determined by FISH. (A) tRNAMet accumulates in the nuclei of cca1–1 cells. Wild-type (SS328; 1, 1′), nup116–5∷HIS3 (SWY27; 2, 2′) or cca1–1 (ts352; 3, 3′), were prepared for FISH and the RNAs were hybridized with tagged oligonucleotides specific to tRNAMet or poly(A) to locate tRNA (1′, 2′, 3′) or mRNAs (1, 2, 3). (B) Overexpressed Cca1p suppresses the tRNA nuclear export defect caused by the los1∷Kanr mutation. los1∷Kanr cells containing YEp24 (1, 1′), YEpCCA1 (2, 2′), YEpLOS1-ET (3, 3′). Cells in 1, 2, and 3 were hybridized with an oligonucleotide specific to tRNAMet; 1′, 2′, and 3′, the same cells stained with 4′,6-diamidino-2-phenylindole.
Figure 2
Figure 2
(A) Overexpressed Cca1p suppresses the los1 growth defect. LOS1, los1–1, and los1∷Kanr cells containing YEp24 or YEpCCA1 were grown to saturation and serially diluted; aliquots were spotted onto solid rich media and incubated at 23°C or 34°C for 3 days. (B) Overexpressed nucleus-located Cca1p suppresses los1. Los1∷Kanr cells containing YEpCCA1, pRS426NLS-CCA1, or pRS426NLS-CCA1-GFP we tested for los1 suppression as in A. (C) pRS426NLS-CCA1 and pRS426NLS-CCA1-GFP encode active Cca1 proteins. LOS1 (designated 1) or cca1–1 (designated 2–5) cells containing pRS426 (designated 1, 2), YEpCCA1 (designated 3), pRS426NLS-CCA1 (designated 4), or pRS426NLS-CCA1-GFP (designated 5) were streaked for single colonies and were grown on rich media at 23°C and 37°C.
Figure 3
Figure 3
Mes1p is a cognate tRNA-specific multicopy suppressor of the los1∷Kanr tRNAMet nuclear export defect. FISH for los1∷Kanr cells containing YEp24 (A-A′ and D-D′), YEpMES1 (B-B′ and E-E′), or YEpLOS1-ET (C-C′ and F-F′). Cells in A–C were hybridized with an oligonucleotide specific to tRNAMet; D–F were hybridized with an oligonucleotide specific for tRNAIleAAU. A′–F′, the same cells stained with 4′,6-diamidino-2-phenylindole.
Figure 4
Figure 4
Multicopy Cca1p does not suppress the los1 pre-tRNA-splicing defect. RNAs were prepared from LOS1 (X2316–3C) and los1∷kanr (SS700) cells containing YEp24, YEpCCA1, or YEpLOS1-ET. The RNAs were transferred to membranes and hybridized with an oligonucleotide specific for tRNAIleUAU. The identity of the initial transcripts, IVS-tRNAs and mature tRNAs are indicated in cartoon form.
Figure 5
Figure 5
Cca1p can shuttle to an introduced nucleus in heterokaryons. Wild-type (SS330) cells containing SV40NLS-CCA1-III-GFP-pRS416GAL1/10F or pGAL-H2B-GFP pregrown in media containing galactose were transferred to media containing glucose and were mated to kar1–1 cells (MS739). After zygotes were formed (≈1.5 h), the locations of the GFP-tagged proteins in live cells were visualized. (Left) A1–A4 and B1–B2, images of NLS-Cca1p-III-GFP and H2B-GFP, respectively, viewed in the GFP channel; (Center) differential interference contrast microscopy of the same cells; (Right) converged GFP/differential interference contrast microscopy images.
Figure 6
Figure 6
Models for tRNA nuclear export (1). Los1p-dependent pathway. It is unknown whether Cca1p and nuclear tRNA aminoacylation function in this pathway in yeast (2). Three possible mechanisms for the Los1p-independent pathway. (2a) Cca1p and nuclear aminoacylation function in series to prepare tRNAs for interaction with an unknown “exportin”; (2b) Cca1p interacts with tRNA, remains bound after catalysis, and ferries tRNA to the cytosol; (2c) a mechanism similar to 2b in which putative shuttling of aaRS serves to export tRNA via stable aaRS/tRNA complexes.
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