tRNA nuclear export in saccharomyces cerevisiae: in situ hybridization analysis

Abstract

To understand the factors specifically affecting tRNA nuclear export, we adapted in situ hybridization procedures to locate endogenous levels of individual tRNA families in wild-type and mutant yeast cells. Our studies of tRNAs encoded by genes lacking introns show that nucleoporin Nup116p affects both poly(A) RNA and tRNA export, whereas Nup159p affects only poly(A) RNA export. Los1p is similar to exportin-t, which facilitates vertebrate tRNA export. A los1 deletion mutation affects tRNA but not poly(A) RNA export. The data support the notion that Los1p and exportin-t are functional homologues. Because LOS1 is nonessential, tRNA export in vertebrate and yeast cells likely involves factors in addition to exportin-t. Mutation of RNA1, which encodes RanGAP, causes nuclear accumulation of tRNAs and poly(A) RNA. Many yeast mutants, including those with the rna1-1 mutation, affect both pre-tRNA splicing and RNA export. Our studies of the location of intron-containing pre-tRNAs in the rna1-1 mutant rule out the possibility that this results from tRNA export occurring before splicing. Our results also argue against inappropriate subnuclear compartmentalization causing defects in pre-tRNA splicing. Rather, the data support "feedback" of nucleus/cytosol exchange to the pre-tRNA splicing machinery.

Figure 1

Diagram of the regions of RNAs complementary to oligonucleotide probes. The straight line depicts mature RNA regions; the wavy line represents the IVS sequence, and the interrupted line marks the position from where an intron has been removed. Lines with arrows indicate the complementary regions of each probe.

Figure 2

Northern blot detection of pre-tRNA^Ile_UAU, mature tRNA^Ile_UAU, and mature tRNA^Ile_AAU. tRNAs were isolated from EE1b-35 (lanes 1, 2, 5, 6, 9, and 10) and EE1b-6 (lanes 3, 4, 7, 8, 11, and 12) cells grown either at 23°C (lanes 1, 3, 5, 7, 9, and 11) or shifted to 37°C for 1 h (lanes 2, 4, 6, 8, 10, and 12). Lanes 1–4 were incubated with probe 04, lanes 5–8 were incubated with probe 05, and lanes 9–11 were incubated first with probe 05 and then stripped and reprobed with probe 03. In lanes 1–4, the top band corresponds to 5′ and 3′ extended intron-containing pre-tRNA species, the next band corresponds to end-matured intron-containing pre-tRNA, and the bottom band corresponds to mature tRNA^Ile_UAU.

Figure 3

Detection of endogenous levels of single species of tRNA is possible by fluorescence in situ hybridization. EE1b-35 cells were grown at 23°C and then subjected to fluorescence in situ hybridization. (A) In situ hybridization done in the absence of digoxigenin-labeled probe. (C) In situ hybridization detection of mature tRNA^Ile_AAU using digoxigenin-labeled probe 05. (E) In situ hybridization detection of mature tRNA^Ile_AAU using digoxigenin-labeled probe 05 in the presence of 1000-fold excess of unlabeled probe 05. (G) In situ hybridization detection of mature tRNA^Ile_UAU using digoxigenin-labeled probe 04. (I) In situ hybridization detection of pre-tRNA^Ile_UAU using digoxigenin-labeled probe 03. (B, D, F, H, and J) DAPI staining of cells shown in A, C, E, G, and I, respectively.

Figure 4

Identification of tRNA transport mutants is possible using fluorescence in situ hybridization. Parent W303 and nup116Δ strain SWY27 were grown at 23°C, and log phase cells were shifted to 37°C for 1 h. (A) In situ hybridization detection of Poly(A) RNA with digoxigenin-labeled probe 02 in W303 cells. (C) Detection of Poly(A) RNA with digoxigenin-labeled probe 02 in SWY27 cells. (E) Detection of mature tRNA^Ile_AAU with digoxigenin-labeled probe 05 in W303 cells. (G) Mature tRNA^Ile_AAU detection with digoxigenin-labeled probe 05 in SWY27 cells. (B, D, F, and H) DAPI staining of cells shown in A, C, E, and G, respectively.

Figure 5

Nucleoporins that are required for Poly(A) RNA export may not be required for mature tRNA export out of the nucleus. Parent FY86 and rat7-1 mutant strain LGY101 were grown at 23°C, and log phase cells were shifted to 37°C for 1 h. In situ hybridization was performed using the following probes and cells: (A) poly(A) RNA probe 02, FY86 cells; (C) poly(A) RNA probe 02, LGY101 cells; (E) mature tRNA^Ile_AAU probe 05, FY86 cells; (G) mature tRNA^Ile_AAU probe 05, LGY101 cells. (B, D, F, and H) DAPI staining of cells shown in A, C, E, and G, respectively.

Figure 6

(A) Deletion of LOS1 affects mature tRNA nuclear export. Parent X2316-3C and los1Δ mutant strain IIId1c-ΔV were grown at 23°C, and log phase cells were shifted to 37°C for 1 h. In situ hybridization was as follows: (panel A) detection of Poly(A) RNA with probe 02 in X2316-3C cells; (panel C) detection of Poly(A) RNA with probe 02 in IIId1c-ΔV cells; (panel E) detection of mature tRNA^Ile_AAU with probe 05 in X2316-3C cells; (panel G) in situ hybridization detection of mature tRNA^Ile_AAU with digoxigenin-labeled probe 05 in IIId1c-ΔV cells. (panels B, D, F, and H) DAPI staining of cells shown in panels A, C, E, and G, respectively. (B) Simultaneous in situ hybridization analysis of tRNA^Ile_AAU and immunofluorescent location of Nsp1. Parent X2316-3C and los1Δ mutant strain IIId1c-ΔV were grown at 23°C and log phase cells were shifted to 37°C for 1 h. (Panel A) Detection of mature tRNA^Ile_AAU with probe 05 in X2316-3C cells. (Panel B) Indirect immunofluorescence detection of nucleoporin Nsp1p with 32D6 antibody in X2316-3C cells. (Panel C) Detection of mature tRNA^Ile_AAU with probe 05 in IIId1c-ΔV cells. (Panel D) Indirect immunofluorescence detection of nucleoporin Nsp1p with 32D6 antibody in IIId1c-ΔV cells. Arrows point to cells displaying nuclear accumulations.

Figure 7

Mutation in RNA1 causes accumulation of mature tRNA in the nucleus. The same cultures of parent EE1b-35 and rna1-1 mutant strain EE1b-6 that were grown at 23°C and used for Figure 3 were shifted to 37°C for 3 h. (A) Detection of Poly(A) RNA with probe 02 in EE1b-35 cells. (C) Detection of Poly(A) RNA with probe 02 in EE1b-6 cells. (E) Detection of mature tRNA^Ile_AAU with probe 05 in EE1b-35 cells. (G) Detection of mature tRNA^Ile_AAU with probe 05 in EE1b-6 cells. (B, D, F, and H) DAPI staining of cells shown in A, C, E, and G, respectively. Arrows point to cells displaying nuclear accumulations.

Figure 8

Accumulation of pre-tRNAs in rna1-1 cells is not due to precocious movement of pre-tRNAs out of the nucleus. Parent EE1b-35 and rna1-1 mutant strain EE1b-6 were grown at 23°C, and log phase cells were shifted to 37°C for 1 h. (A) Detection of pre-tRNA^Ile_UAU with probe 03 in EE1b-35 cells. (C) Detection of pre-tRNA^Ile_UAU with probe 03 in EE1b-6 cells. (B and D) DAPI staining of cells shown in A and C, respectively.