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An exclusively nuclear RNA-binding protein affects asymmetric localization of ASH1 mRNA and Ash1p in yeast.

Long RM, Gu W, Meng X, Gonsalvez G, Singer RH, Chartrand P - J. Cell Biol. (2001)

Bottom Line: LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA.Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm.We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.

View Article: PubMed Central - PubMed

Affiliation: Medical College of Wisconsin, Department of Microbiology and Molecular Genetics, Milwaukee, Wisconsin 53226, USA. rlong@mcw.edu

ABSTRACT
The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3'-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3'-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.

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Intracellular distribution of the Loc1p-myc protein. (A) Immunofluorescence detection of the Loc1p-myc. Strain YLM090 (loc1) was transformed with the plasmid pRL094 (LOC1-MYC). Loc1p-myc (in red) is expressed throughout the cell cycle and appears to be strictly nuclear (see DAPI, in blue). The two panels show cells before (left) and during (right) anaphase. Bar, 10 μm. (B) Shuttling assay for Loc1p-myc protein. (a–f) Npl3p-GFP at 24°C (a–c) and 36°C (d–f). (g–l) Loc1p-myc at 24°C (g–i) and 36°C (j–l). Whereas the Npl3p-GFP (in green) accumulates in the cytoplasm after a temperature shift from 24°C (a) to 36°C (d), Loc1p-myc (in red) remains in the nucleus at 24°C (g) and 36°C (j). g and j are an overlap of images h and i, and k and l, respectively. b, e, i, and l are DAPI staining (in blue) and c and f are Nomarski. Bar, 10 μm. (C) Heterokaryon shuttling assay for Loc1p-myc. (a–c) Loc1p-myc. (d–f) Npl3p-GFP. Binuclear yeasts show no accumulation of Loc1p-myc (in red) in the second nucleus (a), whereas Npl3p-GFP (in green) appears in both nuclei after cell fusion (d). b and e are DAPI staining (in blue) and c and f are Nomarski. Bar, 10 μm.
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Figure 8: Intracellular distribution of the Loc1p-myc protein. (A) Immunofluorescence detection of the Loc1p-myc. Strain YLM090 (loc1) was transformed with the plasmid pRL094 (LOC1-MYC). Loc1p-myc (in red) is expressed throughout the cell cycle and appears to be strictly nuclear (see DAPI, in blue). The two panels show cells before (left) and during (right) anaphase. Bar, 10 μm. (B) Shuttling assay for Loc1p-myc protein. (a–f) Npl3p-GFP at 24°C (a–c) and 36°C (d–f). (g–l) Loc1p-myc at 24°C (g–i) and 36°C (j–l). Whereas the Npl3p-GFP (in green) accumulates in the cytoplasm after a temperature shift from 24°C (a) to 36°C (d), Loc1p-myc (in red) remains in the nucleus at 24°C (g) and 36°C (j). g and j are an overlap of images h and i, and k and l, respectively. b, e, i, and l are DAPI staining (in blue) and c and f are Nomarski. Bar, 10 μm. (C) Heterokaryon shuttling assay for Loc1p-myc. (a–c) Loc1p-myc. (d–f) Npl3p-GFP. Binuclear yeasts show no accumulation of Loc1p-myc (in red) in the second nucleus (a), whereas Npl3p-GFP (in green) appears in both nuclei after cell fusion (d). b and e are DAPI staining (in blue) and c and f are Nomarski. Bar, 10 μm.

Mentions: Surprisingly, we found that Loc1p-myc6 was present only in the nucleus of yeast cells and apparently absent from the cytoplasm, even when overexpressed and imaged with maximal sensitivity (Fig. 8 A). Loc1p-myc was never observed coincident with the mRNA at the bud tip of late anaphase cells. This result suggests that Loc1p is essentially nuclear; however, this experiment did not rule out the possibility that Loc1p could shuttle between the nucleus and the cytoplasm.


An exclusively nuclear RNA-binding protein affects asymmetric localization of ASH1 mRNA and Ash1p in yeast.

Long RM, Gu W, Meng X, Gonsalvez G, Singer RH, Chartrand P - J. Cell Biol. (2001)

Intracellular distribution of the Loc1p-myc protein. (A) Immunofluorescence detection of the Loc1p-myc. Strain YLM090 (loc1) was transformed with the plasmid pRL094 (LOC1-MYC). Loc1p-myc (in red) is expressed throughout the cell cycle and appears to be strictly nuclear (see DAPI, in blue). The two panels show cells before (left) and during (right) anaphase. Bar, 10 μm. (B) Shuttling assay for Loc1p-myc protein. (a–f) Npl3p-GFP at 24°C (a–c) and 36°C (d–f). (g–l) Loc1p-myc at 24°C (g–i) and 36°C (j–l). Whereas the Npl3p-GFP (in green) accumulates in the cytoplasm after a temperature shift from 24°C (a) to 36°C (d), Loc1p-myc (in red) remains in the nucleus at 24°C (g) and 36°C (j). g and j are an overlap of images h and i, and k and l, respectively. b, e, i, and l are DAPI staining (in blue) and c and f are Nomarski. Bar, 10 μm. (C) Heterokaryon shuttling assay for Loc1p-myc. (a–c) Loc1p-myc. (d–f) Npl3p-GFP. Binuclear yeasts show no accumulation of Loc1p-myc (in red) in the second nucleus (a), whereas Npl3p-GFP (in green) appears in both nuclei after cell fusion (d). b and e are DAPI staining (in blue) and c and f are Nomarski. Bar, 10 μm.
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Figure 8: Intracellular distribution of the Loc1p-myc protein. (A) Immunofluorescence detection of the Loc1p-myc. Strain YLM090 (loc1) was transformed with the plasmid pRL094 (LOC1-MYC). Loc1p-myc (in red) is expressed throughout the cell cycle and appears to be strictly nuclear (see DAPI, in blue). The two panels show cells before (left) and during (right) anaphase. Bar, 10 μm. (B) Shuttling assay for Loc1p-myc protein. (a–f) Npl3p-GFP at 24°C (a–c) and 36°C (d–f). (g–l) Loc1p-myc at 24°C (g–i) and 36°C (j–l). Whereas the Npl3p-GFP (in green) accumulates in the cytoplasm after a temperature shift from 24°C (a) to 36°C (d), Loc1p-myc (in red) remains in the nucleus at 24°C (g) and 36°C (j). g and j are an overlap of images h and i, and k and l, respectively. b, e, i, and l are DAPI staining (in blue) and c and f are Nomarski. Bar, 10 μm. (C) Heterokaryon shuttling assay for Loc1p-myc. (a–c) Loc1p-myc. (d–f) Npl3p-GFP. Binuclear yeasts show no accumulation of Loc1p-myc (in red) in the second nucleus (a), whereas Npl3p-GFP (in green) appears in both nuclei after cell fusion (d). b and e are DAPI staining (in blue) and c and f are Nomarski. Bar, 10 μm.
Mentions: Surprisingly, we found that Loc1p-myc6 was present only in the nucleus of yeast cells and apparently absent from the cytoplasm, even when overexpressed and imaged with maximal sensitivity (Fig. 8 A). Loc1p-myc was never observed coincident with the mRNA at the bud tip of late anaphase cells. This result suggests that Loc1p is essentially nuclear; however, this experiment did not rule out the possibility that Loc1p could shuttle between the nucleus and the cytoplasm.

Bottom Line: LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA.Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm.We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.

View Article: PubMed Central - PubMed

Affiliation: Medical College of Wisconsin, Department of Microbiology and Molecular Genetics, Milwaukee, Wisconsin 53226, USA. rlong@mcw.edu

ABSTRACT
The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3'-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3'-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.

Show MeSH
Related in: MedlinePlus