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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 formation of the specific RNA–protein complex is dependent on Loc1p. Band mobility shift assays were performed as mentioned in the legend to Fig. 1. Lane 1, E3 RNA probe only, no protein extract. Lanes 2 and 3, E3 transcripts were incubated with yeast extracts of YLM090 (loc1) and K699 (wild-type) strains. Lane 4, E3 transcripts incubated with extracts of YLM090 (loc1) complemented with a LOC1-myc plasmid.
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Figure 3: The formation of the specific RNA–protein complex is dependent on Loc1p. Band mobility shift assays were performed as mentioned in the legend to Fig. 1. Lane 1, E3 RNA probe only, no protein extract. Lanes 2 and 3, E3 transcripts were incubated with yeast extracts of YLM090 (loc1) and K699 (wild-type) strains. Lane 4, E3 transcripts incubated with extracts of YLM090 (loc1) complemented with a LOC1-myc plasmid.

Mentions: We more closely investigated Loc1p RNA-binding activity by mobility shift assay using E3 RNA probe and extracts prepared from wild-type and loc1 strains (Fig. 3). An RNA–protein complex of the same electrophoretic mobility was not detected from the loc1 extract (Fig. 3, lane 2) compared with the wild-type extract (Fig. 3, lane 3). However, a new complex of lower molecular weight appeared from the loc1 extract, possibly coming from a nonspecific RNA-binding protein. After incubating element E3 with an extract prepared from a loc1 strain expressing Loc1p-myc from a multicopy plasmid (Fig. 3, lane 4), we observed that the specific RNA–protein complex was restored. This result indicates that Loc1p is required for the formation of the higher molecular weight RNA–protein complex. However, these results do not distinguish whether Loc1p is a component of the complex or if Loc1p directly binds element E3.


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 formation of the specific RNA–protein complex is dependent on Loc1p. Band mobility shift assays were performed as mentioned in the legend to Fig. 1. Lane 1, E3 RNA probe only, no protein extract. Lanes 2 and 3, E3 transcripts were incubated with yeast extracts of YLM090 (loc1) and K699 (wild-type) strains. Lane 4, E3 transcripts incubated with extracts of YLM090 (loc1) complemented with a LOC1-myc plasmid.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169461&req=5

Figure 3: The formation of the specific RNA–protein complex is dependent on Loc1p. Band mobility shift assays were performed as mentioned in the legend to Fig. 1. Lane 1, E3 RNA probe only, no protein extract. Lanes 2 and 3, E3 transcripts were incubated with yeast extracts of YLM090 (loc1) and K699 (wild-type) strains. Lane 4, E3 transcripts incubated with extracts of YLM090 (loc1) complemented with a LOC1-myc plasmid.
Mentions: We more closely investigated Loc1p RNA-binding activity by mobility shift assay using E3 RNA probe and extracts prepared from wild-type and loc1 strains (Fig. 3). An RNA–protein complex of the same electrophoretic mobility was not detected from the loc1 extract (Fig. 3, lane 2) compared with the wild-type extract (Fig. 3, lane 3). However, a new complex of lower molecular weight appeared from the loc1 extract, possibly coming from a nonspecific RNA-binding protein. After incubating element E3 with an extract prepared from a loc1 strain expressing Loc1p-myc from a multicopy plasmid (Fig. 3, lane 4), we observed that the specific RNA–protein complex was restored. This result indicates that Loc1p is required for the formation of the higher molecular weight RNA–protein complex. However, these results do not distinguish whether Loc1p is a component of the complex or if Loc1p directly binds element E3.

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