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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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The ASH1 mRNA is delocalized in a loc1 yeast strain. (A) Fluorescent in situ hybridization for ASH1 mRNA in wild-type (K699) and loc1 (YLM090) yeast cells. ASH1 mRNA is expressed from a multicopy plasmid (YEPlac181). DAPI, DNA staining; NOM., Nomarski. The ASH1 mRNA is tightly localized at the bud tip of a wild-type cell, whereas it is delocalized in the cytoplasm of a late anaphase loc1 yeast cell. Bar, 10 μm. (B) Immunofluorescence detection of the Ash1p-myc in postanaphase YML094 (cla4) and YML093 (cla4 loc1) yeast cells. DAPI, DNA staining; NOM., Nomarski. Note the elongated bud neck caused by the deletion of the CLA4 gene. In the majority of cells, Ash1p-myc is symmetrically distributed in the loc1 strain. Bar, 10 μm.
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Figure 7: The ASH1 mRNA is delocalized in a loc1 yeast strain. (A) Fluorescent in situ hybridization for ASH1 mRNA in wild-type (K699) and loc1 (YLM090) yeast cells. ASH1 mRNA is expressed from a multicopy plasmid (YEPlac181). DAPI, DNA staining; NOM., Nomarski. The ASH1 mRNA is tightly localized at the bud tip of a wild-type cell, whereas it is delocalized in the cytoplasm of a late anaphase loc1 yeast cell. Bar, 10 μm. (B) Immunofluorescence detection of the Ash1p-myc in postanaphase YML094 (cla4) and YML093 (cla4 loc1) yeast cells. DAPI, DNA staining; NOM., Nomarski. Note the elongated bud neck caused by the deletion of the CLA4 gene. In the majority of cells, Ash1p-myc is symmetrically distributed in the loc1 strain. Bar, 10 μm.

Mentions: Images were captured using the Esprit Image Analysis software (Life Science Resources) with an OlymPix TE cooled 12-bit CCD camera (Life Science Resources) mounted on an Olympus BX-60 fluorescence microscope (Olympus) with a PlanApo 100×, 1.35 NA objective (Olympus). Single plane images were captured and processed using the Adobe Photoshop 5.0 software (Adobe Systems). For Fig. 7 B, panels g–l, a three-dimensional data set, composed of 30 images separated by 200 nm in the axial direction, was acquired and deconvolved with an acquired point spread function (PSF) using EPR software (Scanalytics). 7 to 10 planes for both Cy3 and DAPI channels were overlapped to give panels h and k in Fig. 7 B using Adobe Photoshop 5.0 software.


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)

The ASH1 mRNA is delocalized in a loc1 yeast strain. (A) Fluorescent in situ hybridization for ASH1 mRNA in wild-type (K699) and loc1 (YLM090) yeast cells. ASH1 mRNA is expressed from a multicopy plasmid (YEPlac181). DAPI, DNA staining; NOM., Nomarski. The ASH1 mRNA is tightly localized at the bud tip of a wild-type cell, whereas it is delocalized in the cytoplasm of a late anaphase loc1 yeast cell. Bar, 10 μm. (B) Immunofluorescence detection of the Ash1p-myc in postanaphase YML094 (cla4) and YML093 (cla4 loc1) yeast cells. DAPI, DNA staining; NOM., Nomarski. Note the elongated bud neck caused by the deletion of the CLA4 gene. In the majority of cells, Ash1p-myc is symmetrically distributed in the loc1 strain. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 7: The ASH1 mRNA is delocalized in a loc1 yeast strain. (A) Fluorescent in situ hybridization for ASH1 mRNA in wild-type (K699) and loc1 (YLM090) yeast cells. ASH1 mRNA is expressed from a multicopy plasmid (YEPlac181). DAPI, DNA staining; NOM., Nomarski. The ASH1 mRNA is tightly localized at the bud tip of a wild-type cell, whereas it is delocalized in the cytoplasm of a late anaphase loc1 yeast cell. Bar, 10 μm. (B) Immunofluorescence detection of the Ash1p-myc in postanaphase YML094 (cla4) and YML093 (cla4 loc1) yeast cells. DAPI, DNA staining; NOM., Nomarski. Note the elongated bud neck caused by the deletion of the CLA4 gene. In the majority of cells, Ash1p-myc is symmetrically distributed in the loc1 strain. Bar, 10 μm.
Mentions: Images were captured using the Esprit Image Analysis software (Life Science Resources) with an OlymPix TE cooled 12-bit CCD camera (Life Science Resources) mounted on an Olympus BX-60 fluorescence microscope (Olympus) with a PlanApo 100×, 1.35 NA objective (Olympus). Single plane images were captured and processed using the Adobe Photoshop 5.0 software (Adobe Systems). For Fig. 7 B, panels g–l, a three-dimensional data set, composed of 30 images separated by 200 nm in the axial direction, was acquired and deconvolved with an acquired point spread function (PSF) using EPR software (Scanalytics). 7 to 10 planes for both Cy3 and DAPI channels were overlapped to give panels h and k in Fig. 7 B using Adobe Photoshop 5.0 software.

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