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Ultrastructural localization of rRNA shows defective nuclear export of preribosomes in mutants of the Nup82p complex.

Gleizes PE, Noaillac-Depeyre J, Léger-Silvestre I, Teulières F, Dauxois JY, Pommet D, Azum-Gelade MC, Gas N - J. Cell Biol. (2001)

Bottom Line: Interestingly, domains of Nup159p required for mRNP trafficking are not necessary for preribosome export.Furthermore, the RNA helicase Dbp5p and the protein Gle1p, which interact with Nup159p and are involved in mRNP trafficking, are dispensable for ribosomal transport.Thus, the Nup82p-Nup159p-Nsp1p nucleoporin complex is part of the nuclear export pathways of preribosomes and mRNPs, but with distinct functions in these two processes.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biologie Moléculaire Eucaryote, Centre Nationale de la Recherche Scientifique and Université Paul Sabatier, 31062 Toulouse cedex, France. gleizes@ibcg.biotoul.fr

ABSTRACT
To study the nuclear export of preribosomes, ribosomal RNAs were detected by in situ hybridization using fluorescence and EM, in the yeast Saccharomyces cerevisiae. In wild-type cells, semiquantitative analysis shows that the distributions of pre-40S and pre-60S particles in the nucleolus and the nucleoplasm are distinct, indicating uncoordinated transport of the two subunits within the nucleus. In cells defective for the activity of the GTPase Gsp1p/Ran, ribosomal precursors accumulate in the whole nucleus. This phenotype is reproduced with pre-60S particles in cells defective in pre-rRNA processing, whereas pre-40S particles only accumulate in the nucleolus, suggesting a tight control of the exit of the small subunit from the nucleolus. Examination of nucleoporin mutants reveals that preribosome nuclear export requires the Nup82p-Nup159p-Nsp1p complex. In contrast, mutations in the nucleoporins forming the Nup84p complex yield very mild or no nuclear accumulation of preribosome. Interestingly, domains of Nup159p required for mRNP trafficking are not necessary for preribosome export. Furthermore, the RNA helicase Dbp5p and the protein Gle1p, which interact with Nup159p and are involved in mRNP trafficking, are dispensable for ribosomal transport. Thus, the Nup82p-Nup159p-Nsp1p nucleoporin complex is part of the nuclear export pathways of preribosomes and mRNPs, but with distinct functions in these two processes.

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Strains defective in pre-rRNA maturation or ribosomal protein trafficking accumulate pre-60S, but not pre-40S particles in the nucleoplasm under nonpermissive conditions. EM in situ hybridization was performed with the 18S, 25S, and ITS2 probes on the pre-rRNA processing mutants gar1-1ts (transferred for 3 h to 37°C) (a–c) and GAL::NOP1 (grown for 20 h with glucose) (d–f). Pre-60S particles are detected in high amount in the whole nucleus, whereas pre-40S particles accumulate in the nucleolus. Nucleolar, but not nucleoplasmic, accumulation of pre-40S particles is also observed in GAL::RRP7 cells cultured with glucose for 16 h (g) and in rps27BΔ cells (h). In the nucleus of pse1-1ts/Δkap123 cells shifted to 37°C for 2 h, pre-rRNAs are found in the nucleolus, when detected with a probe against the entire ribosomal transcription unit (35S probe) (i). Bars, 200 nm.
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fig3: Strains defective in pre-rRNA maturation or ribosomal protein trafficking accumulate pre-60S, but not pre-40S particles in the nucleoplasm under nonpermissive conditions. EM in situ hybridization was performed with the 18S, 25S, and ITS2 probes on the pre-rRNA processing mutants gar1-1ts (transferred for 3 h to 37°C) (a–c) and GAL::NOP1 (grown for 20 h with glucose) (d–f). Pre-60S particles are detected in high amount in the whole nucleus, whereas pre-40S particles accumulate in the nucleolus. Nucleolar, but not nucleoplasmic, accumulation of pre-40S particles is also observed in GAL::RRP7 cells cultured with glucose for 16 h (g) and in rps27BΔ cells (h). In the nucleus of pse1-1ts/Δkap123 cells shifted to 37°C for 2 h, pre-rRNAs are found in the nucleolus, when detected with a probe against the entire ribosomal transcription unit (35S probe) (i). Bars, 200 nm.

Mentions: Although accumulation of preribosomes in the whole nucleus is expected from an alteration of the export machinery per se (namely the NPC, karyopherins, and Ran), such a phenotype could also result from the release from the nucleolus of aborted preribosomes incompetent for nuclear export (due to a defect in pre-rRNA processing or in the assembly of a subunit), or from the failure of a maturation step that normally occurs in the nucleoplasm. For example, we have shown previously that preribosomes strongly accumulate in the nucleus of cells bearing a mutated 5S rRNA (Dechampesme et al., 1999). To gain more insight into the fate of preribosomes upon a defect in pre-rRNA processing, we looked at strains bearing conditional mutations of NOP1 and GAR1, two genes encoding nucleolar proteins found in C + D and H/ACA box snoRNPs, respectively. Depletion of Nop1p in a GAL::NOP1 strain leads to a general defect in pre-rRNA processing and methylation (Tollervey et al., 1991). The ts gar1-1 mutant is mostly affected in the synthesis of 18S rRNA, and pre-rRNA pseudouridylation is abolished (Bousquet-Antonelli et al., 1997). Observation of these mutants under nonpermissive conditions revealed different accumulation patterns for the small and large subunits (Fig. 3 , a–f). The nucleolar labeling was higher with both probes (18S and 25S) under nonpermissive conditions, probably due to accumulation of ill-processed pre-rRNAs. In addition, the amount pre-60S particles dramatically increased in the nucleoplasm in both gar1-1 and GAL::NOP1 mutants. The ITS2 probe revealed a large amount of pre-25S/pre-5.8S rRNA in the nucleoplasm of these cells, indicating the release of immature pre-60S particles from the nucleolus (compare with wild-type cells in Fig. 1). Although these cells were shown to accumulate aberrant precursors (23S) of the 18S rRNA, the loss of Gar1p or Nop1p function did not result in the accumulation of 18S precursors in the nucleoplasm. Because Nop1p or Gar1p act in early steps of pre-rRNA processing, we studied a strain conditionally expressing RRP7 (GAL::RRP7), an essential gene whose product is required downstream of Nop1p and Gar1p in the synthesis of the small subunit (Baudin-Baillieu et al., 1997). Upon depletion of Rrp7p, no nucleoplasmic accumulation of pre-18S rRNA was observed (Fig. 3 g); a similar result was obtained with a mutant deleted of the ribosomal protein gene RPS27B (Fig. 3 h), which can act as a multicopy suppressor of RRP7 deletion, and whose absence severely impairs growth and 18S rRNA production (Baudin-Baillieu et al., 1997). Last, in a double mutant of Kap121p/Pse1p and Kap123p (pse1-1Δkap123), two karyopherins involved in import of ribosomal proteins (Rout et al., 1997), (pre-) rRNAs accumulated in the nucleolus but not in the nucleoplasm, as checked with a probe to the entire ribosomal transcription unit (Fig. 3 i).


Ultrastructural localization of rRNA shows defective nuclear export of preribosomes in mutants of the Nup82p complex.

Gleizes PE, Noaillac-Depeyre J, Léger-Silvestre I, Teulières F, Dauxois JY, Pommet D, Azum-Gelade MC, Gas N - J. Cell Biol. (2001)

Strains defective in pre-rRNA maturation or ribosomal protein trafficking accumulate pre-60S, but not pre-40S particles in the nucleoplasm under nonpermissive conditions. EM in situ hybridization was performed with the 18S, 25S, and ITS2 probes on the pre-rRNA processing mutants gar1-1ts (transferred for 3 h to 37°C) (a–c) and GAL::NOP1 (grown for 20 h with glucose) (d–f). Pre-60S particles are detected in high amount in the whole nucleus, whereas pre-40S particles accumulate in the nucleolus. Nucleolar, but not nucleoplasmic, accumulation of pre-40S particles is also observed in GAL::RRP7 cells cultured with glucose for 16 h (g) and in rps27BΔ cells (h). In the nucleus of pse1-1ts/Δkap123 cells shifted to 37°C for 2 h, pre-rRNAs are found in the nucleolus, when detected with a probe against the entire ribosomal transcription unit (35S probe) (i). Bars, 200 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2150900&req=5

fig3: Strains defective in pre-rRNA maturation or ribosomal protein trafficking accumulate pre-60S, but not pre-40S particles in the nucleoplasm under nonpermissive conditions. EM in situ hybridization was performed with the 18S, 25S, and ITS2 probes on the pre-rRNA processing mutants gar1-1ts (transferred for 3 h to 37°C) (a–c) and GAL::NOP1 (grown for 20 h with glucose) (d–f). Pre-60S particles are detected in high amount in the whole nucleus, whereas pre-40S particles accumulate in the nucleolus. Nucleolar, but not nucleoplasmic, accumulation of pre-40S particles is also observed in GAL::RRP7 cells cultured with glucose for 16 h (g) and in rps27BΔ cells (h). In the nucleus of pse1-1ts/Δkap123 cells shifted to 37°C for 2 h, pre-rRNAs are found in the nucleolus, when detected with a probe against the entire ribosomal transcription unit (35S probe) (i). Bars, 200 nm.
Mentions: Although accumulation of preribosomes in the whole nucleus is expected from an alteration of the export machinery per se (namely the NPC, karyopherins, and Ran), such a phenotype could also result from the release from the nucleolus of aborted preribosomes incompetent for nuclear export (due to a defect in pre-rRNA processing or in the assembly of a subunit), or from the failure of a maturation step that normally occurs in the nucleoplasm. For example, we have shown previously that preribosomes strongly accumulate in the nucleus of cells bearing a mutated 5S rRNA (Dechampesme et al., 1999). To gain more insight into the fate of preribosomes upon a defect in pre-rRNA processing, we looked at strains bearing conditional mutations of NOP1 and GAR1, two genes encoding nucleolar proteins found in C + D and H/ACA box snoRNPs, respectively. Depletion of Nop1p in a GAL::NOP1 strain leads to a general defect in pre-rRNA processing and methylation (Tollervey et al., 1991). The ts gar1-1 mutant is mostly affected in the synthesis of 18S rRNA, and pre-rRNA pseudouridylation is abolished (Bousquet-Antonelli et al., 1997). Observation of these mutants under nonpermissive conditions revealed different accumulation patterns for the small and large subunits (Fig. 3 , a–f). The nucleolar labeling was higher with both probes (18S and 25S) under nonpermissive conditions, probably due to accumulation of ill-processed pre-rRNAs. In addition, the amount pre-60S particles dramatically increased in the nucleoplasm in both gar1-1 and GAL::NOP1 mutants. The ITS2 probe revealed a large amount of pre-25S/pre-5.8S rRNA in the nucleoplasm of these cells, indicating the release of immature pre-60S particles from the nucleolus (compare with wild-type cells in Fig. 1). Although these cells were shown to accumulate aberrant precursors (23S) of the 18S rRNA, the loss of Gar1p or Nop1p function did not result in the accumulation of 18S precursors in the nucleoplasm. Because Nop1p or Gar1p act in early steps of pre-rRNA processing, we studied a strain conditionally expressing RRP7 (GAL::RRP7), an essential gene whose product is required downstream of Nop1p and Gar1p in the synthesis of the small subunit (Baudin-Baillieu et al., 1997). Upon depletion of Rrp7p, no nucleoplasmic accumulation of pre-18S rRNA was observed (Fig. 3 g); a similar result was obtained with a mutant deleted of the ribosomal protein gene RPS27B (Fig. 3 h), which can act as a multicopy suppressor of RRP7 deletion, and whose absence severely impairs growth and 18S rRNA production (Baudin-Baillieu et al., 1997). Last, in a double mutant of Kap121p/Pse1p and Kap123p (pse1-1Δkap123), two karyopherins involved in import of ribosomal proteins (Rout et al., 1997), (pre-) rRNAs accumulated in the nucleolus but not in the nucleoplasm, as checked with a probe to the entire ribosomal transcription unit (Fig. 3 i).

Bottom Line: Interestingly, domains of Nup159p required for mRNP trafficking are not necessary for preribosome export.Furthermore, the RNA helicase Dbp5p and the protein Gle1p, which interact with Nup159p and are involved in mRNP trafficking, are dispensable for ribosomal transport.Thus, the Nup82p-Nup159p-Nsp1p nucleoporin complex is part of the nuclear export pathways of preribosomes and mRNPs, but with distinct functions in these two processes.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biologie Moléculaire Eucaryote, Centre Nationale de la Recherche Scientifique and Université Paul Sabatier, 31062 Toulouse cedex, France. gleizes@ibcg.biotoul.fr

ABSTRACT
To study the nuclear export of preribosomes, ribosomal RNAs were detected by in situ hybridization using fluorescence and EM, in the yeast Saccharomyces cerevisiae. In wild-type cells, semiquantitative analysis shows that the distributions of pre-40S and pre-60S particles in the nucleolus and the nucleoplasm are distinct, indicating uncoordinated transport of the two subunits within the nucleus. In cells defective for the activity of the GTPase Gsp1p/Ran, ribosomal precursors accumulate in the whole nucleus. This phenotype is reproduced with pre-60S particles in cells defective in pre-rRNA processing, whereas pre-40S particles only accumulate in the nucleolus, suggesting a tight control of the exit of the small subunit from the nucleolus. Examination of nucleoporin mutants reveals that preribosome nuclear export requires the Nup82p-Nup159p-Nsp1p complex. In contrast, mutations in the nucleoporins forming the Nup84p complex yield very mild or no nuclear accumulation of preribosome. Interestingly, domains of Nup159p required for mRNP trafficking are not necessary for preribosome export. Furthermore, the RNA helicase Dbp5p and the protein Gle1p, which interact with Nup159p and are involved in mRNP trafficking, are dispensable for ribosomal transport. Thus, the Nup82p-Nup159p-Nsp1p nucleoporin complex is part of the nuclear export pathways of preribosomes and mRNPs, but with distinct functions in these two processes.

Show MeSH
Related in: MedlinePlus