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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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Amino acid sequence of Loc1p as predicted from the Yeast Genome Sequencing project. One remarkable feature of Loc1p is the abundance of arginine and lysine in this protein. Loc1p contains 20.1% lysine and 9.8% arginine. The pI value of 10.79 for Loc1p is a reflection of the abundance of positive charge in this protein.
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Figure 2: Amino acid sequence of Loc1p as predicted from the Yeast Genome Sequencing project. One remarkable feature of Loc1p is the abundance of arginine and lysine in this protein. Loc1p contains 20.1% lysine and 9.8% arginine. The pI value of 10.79 for Loc1p is a reflection of the abundance of positive charge in this protein.

Mentions: A plasmid expressing a fusion RNA containing two copies of the MS2 coat protein stem-loop motif and one copy of the ASH1 3′-UTR localization element (E3) was transformed into the three-hybrid host yeast strain, L40-coat. The yeast strain expressing the fusion RNA was transformed with a yeast cDNA library fused to the Gal4p activation domain, and the cells were plated onto selection media. From the 2,000 colonies that grew on the selection plates, only four colonies were found to express the reporter genes dependent on the fusion RNA. The identity of the cDNA inserts was determined by DNA sequencing. From the S. cerevisiae genome database, two cDNAs corresponded to the yeast ribosomal protein L9B/L9A whereas the remaining two cDNAs corresponded to the uncharacterized open reading frame YFR001w (Fig. 2). We have given YFR001w the name LOC1. LOC1 is predicted to encode a 26-kD protein rich in arginine and lysine. It does not demonstrate any homology to known proteins.


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)

Amino acid sequence of Loc1p as predicted from the Yeast Genome Sequencing project. One remarkable feature of Loc1p is the abundance of arginine and lysine in this protein. Loc1p contains 20.1% lysine and 9.8% arginine. The pI value of 10.79 for Loc1p is a reflection of the abundance of positive charge in this protein.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Amino acid sequence of Loc1p as predicted from the Yeast Genome Sequencing project. One remarkable feature of Loc1p is the abundance of arginine and lysine in this protein. Loc1p contains 20.1% lysine and 9.8% arginine. The pI value of 10.79 for Loc1p is a reflection of the abundance of positive charge in this protein.
Mentions: A plasmid expressing a fusion RNA containing two copies of the MS2 coat protein stem-loop motif and one copy of the ASH1 3′-UTR localization element (E3) was transformed into the three-hybrid host yeast strain, L40-coat. The yeast strain expressing the fusion RNA was transformed with a yeast cDNA library fused to the Gal4p activation domain, and the cells were plated onto selection media. From the 2,000 colonies that grew on the selection plates, only four colonies were found to express the reporter genes dependent on the fusion RNA. The identity of the cDNA inserts was determined by DNA sequencing. From the S. cerevisiae genome database, two cDNAs corresponded to the yeast ribosomal protein L9B/L9A whereas the remaining two cDNAs corresponded to the uncharacterized open reading frame YFR001w (Fig. 2). We have given YFR001w the name LOC1. LOC1 is predicted to encode a 26-kD protein rich in arginine and lysine. It does not demonstrate any homology to known proteins.

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